The invention discloses a fluorescence sensing method and kit for simultaneously detecting cortisol, serum testosterone and creatine kinase isoenzyme, and belongs to the field of biomarker detection. The fluorescence sensing method comprises the following steps: (1), preparing a magnetic bead-aptamer-complementary chain compound; (2), preparing DNA tetrahedral tweezers; (3), performing competition between the target object and the complementary chain; (4), carrying out EXPAR amplification reaction; (5), carrying out complementary chain-DNA tweezers reaction; and (6), performing reading: measuring the fluorescence value of the reaction system. The EXPAR isothermal amplification technology is utilized, a complex temperature change process is not needed, stability is good, sensitivity is high, compared with other detection methods, the content of three target substances including testosterone, cortisol and creatine kinase isoenzyme in serum can be detected at the same time, the content of the target substances is detected according to the fluorescence recovery degree, cost is low. And the detection limits can respectively reach 41pM, 68pM and 8pM.