A multiplex PCR amplification method for identifying an unknown biological sample suspected to be from human, which can determine in a single assay whether the unknown biological sample is a human-derived sample or a human-derived sample blended with components of other species; if the sample is (or does contain) a human-derived sample, it will be individually identified synchronously; and the method comprises the steps of: 1) in an unknown biological sample that has been collected, wherein the sample is suspected to be from human; 2) adding the unknown biological sample to be detected directly, or the nucleic acids extracted from the unknown biological sample to be detected, into premixed PCR reagents; 3) performing multiplex PCR amplification by running PCR amplification procedures; 4) detecting and analyzing the PCR amplification products; wherein the premixed PCR reagents comprise primers specific for human DNA genetic markers and primers specific for nuclear genes on the chromosomes of non-human species; wherein the nuclear gene on the chromosomes of non-human species is housekeeping gene ATP5B; wherein the primers comprised in the premixed PCR reagents have the following sequences: Amel: SEQ ID NO: 1-2; D8S1179: SEQ ID NO: 3-4; D21S11: SEQ ID NO: 5-6; D18S51: SEQ ID NO: 7-8; D2S1338: SEQ ID NO: 9-10; Pig ATP5B: SEQ ID NO: 11-12; DuckATP5B: SEQ ID NO: 13-14; D2S441: SEQ ID NO: 15-16; D5S818: SEQ ID NO: 17-18; D7S820: SEQ ID NO: 19-20; D6S1043: SEQ ID NO: 21-22; Penta D: SEQ ID NO: 23-24; Goose ATP5B: SEQ ID NO: 25-26; D3S1358: SEQ ID NO: 27-28; TH01: SEQ ID NO: 29-30; D19S433: SEQ ID NO: 31-32; D12S391: SEQ ID NO: 33-34; Chicken ATP5B: SEQ ID NO: 35-36; DYS391: SEQ ID NO: 37-38; TPOX: SEQ ID NO: 39-40; D16S539: SEQ ID NO: 41-42; D13S317: SEQ ID NO: 43-44; FGA: SEQ ID NO: 45-46; CSF 1PO: SEQ ID NO: 47-48; vWA: SEQ ID NO: 49-50; D1S1656: SEQ ID NO: 51-52; Penta E: SEQ ID NO: 53-54. 1) in an unknown biological sample that has been collected, wherein the sample is suspected to be from human; 2) adding the unknown biological sample to be detected directly, or the nucleic acids extracted from the unknown biological sample to be detected, into premixed PCR reagents; 3) performing multiplex PCR amplification by running PCR amplification procedures; 4) detecting and analyzing the PCR amplification products; wherein the premixed PCR reagents comprise primers specific for human DNA genetic markers and primers specific for nuclear genes on the chromosomes of non-human species; wherein the nuclear gene on the chromosomes of non-human species is housekeeping gene ATP5B; wherein the primers comprised in the premixed PCR reagents have the following sequences: Amel: SEQ ID NO: 1-2; D8S1179: SEQ ID NO: 3-4; D21S11: SEQ ID NO: 5-6; D18S51: SEQ ID NO: 7-8; D2S1338: SEQ ID NO: 9-10; Pig ATP5B: SEQ ID NO: 11-12; DuckATP5B: SEQ ID NO: 13-14; D2S441: SEQ ID NO: 15-16; D5S818: SEQ ID NO: 17-18; D7S820: SEQ ID NO: 19-20; D6S1043: SEQ ID NO: 21-22; Penta D: SEQ ID NO: 23-24; Goose ATP5B: SEQ ID NO: 25-26; D3S1358: SEQ ID NO: 27-28; TH01: SEQ ID NO: 29-30; D19S433: SEQ ID NO: 31-32; D12S391: SEQ ID NO: 33-34; Chicken ATP5B: SEQ ID NO: 35-36; DYS391: SEQ ID NO: 37-38; TPOX: SEQ ID NO: 39-40; D16S539: SEQ ID NO: 41-42; D13S317: SEQ ID NO: 43-44; FGA: SEQ ID NO: 45-46; CSF 1PO: SEQ ID NO: 47-48; vWA: SEQ ID NO: 49-50; D1S1656: SEQ ID NO: 51-52; Penta E: SEQ ID NO: 53-54. wherein the premixed PCR reagents comprise primers specific for human DNA genetic markers and primers specific for nuclear genes on the chromosomes of non-human species; wherein the nuclear gene on the chromosomes of non-human species is housekeeping gene ATP5B; wherein the primers comprised in the premixed PCR reagents have the following sequences: Amel: SEQ ID NO: 1-2; D8S1179: SEQ ID NO: 3-4; D21S11: SEQ ID NO: 5-6; D18S51: SEQ ID NO: 7-8; D2S1338: SEQ ID NO: 9-10; Pig ATP5B: SEQ ID NO: 11-12; DuckATP5B: SEQ ID NO: 13-14; D2S441: SEQ ID NO: 15-16; D5S818: SEQ ID NO: 17-18; D7S820: SEQ ID NO: 19-20; D6S1043: SEQ ID NO: 21-22; Penta D: SEQ ID NO: 23-24; Goose ATP5B: SEQ ID NO: 25-26; D3S1358: SEQ ID NO: 27-28; TH01: SEQ ID NO: 29-30; D19S433: SEQ ID NO: 31-32; D12S391: SEQ ID NO: 33-34; Chicken ATP5B: SEQ ID NO: 35-36; DYS391: SEQ ID NO: 37-38; TPOX: SEQ ID NO: 39-40; D16S539: SEQ ID NO: 41-42; D13S317: SEQ ID NO: 43-44; FGA: SEQ ID NO: 45-46; CSF 1PO: SEQ ID NO: 47-48; vWA: SEQ ID NO: 49-50; D1S1656: SEQ ID NO: 51-52; Penta E: SEQ ID NO: 53-54. Amel: SEQ ID NO: 1-2; D8S1179: SEQ ID NO: 3-4; D21S11: SEQ ID NO: 5-6; D18S51: SEQ ID NO: 7-8; D2S1338: SEQ ID NO: 9-10; Pig ATP5B: SEQ ID NO: 11-12; DuckATP5B: SEQ ID NO: 13-14; D2S441: SEQ ID NO: 15-16; D5S818: SEQ ID NO: 17-18; D7S820: SEQ ID NO: 19-20; D6S1043: SEQ ID NO: 21-22; Penta D: SEQ ID NO: 23-24; Goose ATP5B: SEQ ID NO: 25-26; D3S1358: SEQ ID NO: 27-28; TH01: SEQ ID NO: 29-30; D19S433: SEQ ID NO: 31-32; D12S391: SEQ ID NO: 33-34; Chicken ATP5B: SEQ ID NO: 35-36; DYS391: SEQ ID NO: 37-38; TPOX: SEQ ID NO: 39-40; D16S539: SEQ ID NO: 41-42; D13S317: SEQ ID NO: 43-44; FGA: SEQ ID NO: 45-46; CSF 1PO: SEQ ID NO: 47-48; vWA: SEQ ID NO: 49-50; D1S1656: SEQ ID NO: 51-52; Penta E: SEQ ID NO: 53-54.