A method for preparation of 3D brain organoids comprising: dissociating neurospheres obtained by the RONA method into single cells by accutase, plating on a cell culture plate after counting the cells, and culturing in medium A for 7 days; culturing the neurospheres in medium B until day 25~35, and encapsulating the neurospheres; culturing the neurospheres in medium B until day 55~65, followed by encapsulating said neurospheres for the second time and culturing continually afterwards; wherein medium A comprises retinoic acid, BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium and B27 supplement; wherein medium B comprises: BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium and B27 supplement; and wherein the RONA method comprises the following steps: a) growing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in suspension in human ESC medium without FGF2 for 2 days; b)from days 2 to 6, adding 50 ng/ml of noggin or 1 μM of dorsomorphin and 10 μM of SB431542 to the medium to obtain embyroid bodies (EBs); c) on day 7, transferring free-floating EBs to Matrigel- or laminin-precoated culture plates comprising N2 induction medium to allow the complete attachment of EB aggregates, and continuing the culture to allow the formation of highly compact 3D column-like neural aggregates (RONA), wherein the N2 induction medium comprises DMEM/F12, 1% N2 induction supplement, 100 μM MEM non-essential amino acid solution, 1mM GlutaMAX and 2 μg/ml heparin; and d) collecting RONAs on day 17 and maintaining RONAs as neurospheres in suspension in Neurobasal medium containing B27 minus VitA and 1 mM GlutaMAX for 1 day. dissociating neurospheres obtained by the RONA method into single cells by accutase, plating on a cell culture plate after counting the cells, and culturing in medium A for 7 days; culturing the neurospheres in medium B until day 25~35, and encapsulating the neurospheres; culturing the neurospheres in medium B until day 55~65, followed by encapsulating said neurospheres for the second time and culturing continually afterwards; wherein medium A comprises retinoic acid, BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium and B27 supplement; wherein medium B comprises: BDNF, GDNF, ascorbic acid, cAMP, Neurobasal medium and B27 supplement; and wherein the RONA method comprises the following steps: a) growing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in suspension in human ESC medium without FGF2 for 2 days; b)from days 2 to 6, adding 50 ng/ml of noggin or 1 μM of dorsomorphin and 10 μM of SB431542 to the medium to obtain embyroid bodies (EBs); c) on day 7, transferring free-floating EBs to Matrigel- or laminin-precoated culture plates comprising N2 induction medium to allow the complete attachment of EB aggregates, and continuing the culture to allow the formation of highly compact 3D column-like neural aggregates (RONA), wherein the N2 induction medium comprises DMEM/F12, 1% N2 induction supplement, 100 μM MEM non-essential amino acid solution, 1mM GlutaMAX and 2 μg/ml heparin; and d) collecting RONAs on day 17 and maintaining RONAs as neurospheres in suspension in Neurobasal medium containing B27 minus VitA and 1 mM GlutaMAX for 1 day. a) growing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in suspension in human ESC medium without FGF2 for 2 days; b)from days 2 to 6, adding 50 ng/ml of noggin or 1 μM of dorsomorphin and 10 μM of SB431542 to the medium to obtain embyroid bodies (EBs); c) on day 7, transferring free-floating EBs to Matrigel- or laminin-precoated culture plates comprising N2 induction medium to allow the complete attachment of EB aggregates, and continuing the culture to allow the formation of highly compact 3D column-like neural aggregates (RONA), wherein the N2 induction medium comprises DMEM/F12, 1% N2 induction supplement, 100 μM MEM non-essential amino acid solution, 1mM GlutaMAX and 2 μg/ml heparin; and d) collecting RONAs on day 17 and maintaining RONAs as neurospheres in suspension in Neurobasal medium containing B27 minus VitA and 1 mM GlutaMAX for 1 day.