OBJECTIVE This study reviewed acridine orange fluorescence (AO-F) in exfoliative urinary cytology results of 1,016 inpatients with urothelial cell carcinoma of the bladder and 804 outpatients to investigate the value of AO-F in the diagnosis of bladder cancer. METHODS A total of 1,016 bladder cancer inpatients from October 1995 to October 2005 and 804 outpatients from January 2004 to January 2006 were enrolled in this study. Each patient provided the morning urine specimen of 30-50 ml in a sterile container. Urine sediments were stained by acridine orange and observed with a fluorescence microscope; 60 bladder cancer inpatients from January 2006 to July 2007 were also chosen for the control study of three different detection methods, including AO-F, hematoxylin and eosin and Feulgen staining. RESULTS Of the 1,016 bladder carcinoma samples analyzed, 793 were AO-F positive. Total positive rate of AO-F was 78.05 %. The positive rate was 74.69 % (611/818) for non-muscle invasive bladder carcinoma and 91.91 % (182/198) for muscle invasive bladder carcinoma. A significant correlation of AO-F positivity with clinical stage was observed (P < 0.01). The positive rates among various pathological grades were 66.7 % (32/48) for G1, 67.5 % (319/474) for G2 and 90.4 % (413/457) for G3 with significant differences (P < 0.01). For the 804 outpatients, the sensitivity and specificity of bladder carcinoma were 77.11 and 85.29 %, respectively. CONCLUSIONS With its high sensitivity and specificity, AO-F is superior to other detection methods for bladder carcinoma detection. In addition, it is familiar, non-invasive, quick, cheap and easily repeatable.