The invention discloses a method capable of screening and comparing bioequivalence of multiple human-derived stem cell secretion factors and application thereof. A detection method capable of screening and comparing bioequivalence of multiple human-derived stem cell secretion factors comprises the following steps: culturing Balb/c3t3 cells respectively by using reagents to be detected and a positive control system, and detecting proliferation rate of the Balb/c3t3 cells. The positive control system is established by steps of concentrating and adjusting concentration; the method is capable of concentrating the stem cell growth factors to 1000-3000 times; then the technical scheme is capable of simultaneously screening multiple objects to be detected in batches; the detection cost can be reduced; the method can be applied to the field of screening of formulations of cosmetics and biological medicines, and is capable of simplifying the detection steps, improving the detection efficiency and reducing the detection cost.