A method of preparing a diene-based rubber latex, the method comprising: a polymerization step of polymerizing a conjugated diene based monomer, a crosslinking agent, an emulsifier, and a molecular weight regulator, the step of the polymerization comprising a polymerization initiation step of initiating polymerization of a) 60 to 75 parts by weight of the conjugated diene based monomer, 0.05 to 3 parts by weight of the emulsifier, and 0.01 to 1 part by weight of an initiator at 60 to 75 °C, wherein the emulsifier is added when a polymerization conversion rate is 30% or less; b) a secondary emulsifier addition step of adding 10 to 25 parts by weight of the conjugated diene based monomer and 0.1 to 1.0 part by weight of a secondary emulsifier when a polymerization conversion rate is 30 to 40 %; c) an additional emulsifier addition step of adding 5 to 20 parts by weight of the conjugated diene based monomer and 0.01 to 0.5 parts by weight of the emulsifier when a polymerization conversion rate is 50 to 85 %; and d) a latex obtainment step of terminating polymerization when a polymerization conversion rate is 90 to 99 % and obtaining a diene-based rubber latex, wherein the crosslinking agent is represented by Formula 1 below and is added in an amount of 0.05 to 0.5 parts by weight before initiation of the polymerization or when a polymerization conversion rate is 30 % or less: [Formula 1] [CH 2 =CR 1 CO 2 (R 2 O) n R 3] m C(R 4) l wherein R 1 is hydrogen or a C1 to C10 alkyl group, R2 is a C1 to C10 alkylene group, R3 is a C1 to C10 alkylene group, R4 is a C1 to C10 alkyl group, 1 is an integer of 0 to 2, n is an integer of 4 to 20, m is an integer of 2 to 4, and m + 1 = 4, and the emulsifier comprises a multimeric acid of an unsaturated fatty acid or a metal salt thereof, and the molecular weight regulator is added in an amount of 0.01 to 0.25 parts by weight when a polymerization conversion rate is 40 to 75 %. a) 60 to 75 parts by weight of the conjugated diene based monomer, 0.05 to 3 parts by weight of the emulsifier, and 0.01 to 1 part by weight of an initiator at 60 to 75 °C, wherein the emulsifier is added when a polymerization conversion rate is 30% or less; b) a secondary emulsifier addition step of adding 10 to 25 parts by weight of the conjugated diene based monomer and 0.1 to 1.0 part by weight of a secondary emulsifier when a polymerization conversion rate is 30 to 40 %; c) an additional emulsifier addition step of adding 5 to 20 parts by weight of the conjugated diene based monomer and 0.01 to 0.5 parts by weight of the emulsifier when a polymerization conversion rate is 50 to 85 %; and d) a latex obtainment step of terminating polymerization when a polymerization conversion rate is 90 to 99 % and obtaining a diene-based rubber latex, wherein R 1 is hydrogen or a C1 to C10 alkyl group, R2 is a C1 to C10 alkylene group, R3 is a C1 to C10 alkylene group, R4 is a C1 to C10 alkyl group, 1 is an integer of 0 to 2, n is an integer of 4 to 20, m is an integer of 2 to 4, and m + 1 = 4, and the emulsifier comprises a multimeric acid of an unsaturated fatty acid or a metal salt thereof, and the molecular weight regulator is added in an amount of 0.01 to 0.25 parts by weight when a polymerization conversion rate is 40 to 75 %.