Dipeptide peptidase IV (DPPIV) and fibroblast activation protein (FAP) are isoenzymes. Evidence shows that DPPIV is related to antitumor immunity, and FAP may be a drug target in cancer therapy, making it seem that the two enzymes might have a synergistic role during the proliferation of cancer cells. Surprisingly, herein, we find an adverse action of DPPIV and FAP in the proliferation process by analyzing their changes with two tailor-made ultrasensitive fluorescent probes. First, the up-regulation of DPPIV and down-regulation of FAP in cancer cells under the stimulation of genistein are detected. Then, we find that MGC803 cells with a higher FAP but lower DPPIV level than SGC7901 cells exhibit a faster proliferation rate. Importantly, inhibiting the DPPIV expression with siRNA increases the proliferation rate of MGC803 cells, whereas the FAP inhibition decreases the rate. These findings suggest that the two enzymes play an adverse role during the proliferation of cancer cells, which provides us a new viewpoint for cancer studies.