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Objective To evaluate the application value of multicolor fluorescence in situ hybridization (M-FISH) in postoperative follow-up monitoring of bladder cancer patients with negative urine cytology. Methods A total of 78 patients with urothelial cancer who were treated by transurethral resection of the bladder were included in this study. All of them had negative urine cytology. The cystoscopy and M-FISH were used to monitor the recurrence of the disease , and the results were compared. Results In 76 patients, 14 cases recurred (18.4%). M-FISH only detected 3 cases of recurrence (4%), and 11 cases of missed diagnosis (14.5%). Cystoscopy showed 54 cases with negative results , 1 case of recurrence (low level), and the M-FISH showed positive results. Cystoscopy showed 12 cases were suspicious and 4 cases of recurrence (2 cases of low level, 2 cases of high level), and M-FISH showed 1 case with positive results (low level). Cystoscopy showed 10 cases with positive results, 9 cases of recurrence (5 cases of low level, 4 cases of high level) , and M-FISH showed 1 case with positive results (high level). For that cystoscopy showed negative results , M-FISH showed positive results, the sensitivity of predicting recurrence was 100%, the specificity was 94.3%, the positive predictive value was 25%, and the negative predictive value was 100%. For that cystoscopy showed suspicious results, M-FISH showed positive results, the sensitivity of predicting recurrence was 25%, the specificity was 87.5%, the positive predictive value was 50%, and the negative predictive value was 70%. For that cystoscopy showed positive results, M-FISH showed positive results, the sensitivity of predicting recurrence was 11.1%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 11.1%. Conclusions M-FISH assay in postoperative follow-up monitoring of bladder cancer patients with negative urine cytology had a low detection rate and low economic benefits. It was not considered as a follow-up routine monitoring project in bladder cancer surveillance after transurethral resection. [ABSTRACT FROM AUTHOR]