The invention discloses a method for isolating recombinant human nerve growth factor from mammalian cell culture, comprising the steps of carrying out strong cation exchange chromatography, to be specific, sampling supernate of clarified mammalian cell culture to strong cation exchange chromatography resin, eluting with elution buffer to obtain first recombinant nerve growth factor; carrying out hydrophobic chromatography, to be specific, treating the first recombinant nerve growth factor, sampling to hydrophobic chromatography resin, and eluting with elution buffer to obtain second recombinant nerve growth factor. The method has few operation steps, the production cycle is shortened, recovery rate is increased, the purity is high, the uniformity is good, and in-vitro bioactivity is very high, generally higher than 1.5*105 U / mg, which is far higher than that (0.5*105 U / mg) of mouse nerve growth factors for injection in the market.