Objective To investigate the value of the application of the fresh first morning midstream urine in cytological study of bladder cancer patients. Methods The results of the fresh first and second morning midstream urine cytological studies for 52 bladder cancer patients were analyzed.Continual three urine samples and single urine sample were treated as study objects respectively. The positive rates in different tumor stages and grades were evaluated. Results The positive rate of overall 52 patients was 78. 8 % (41/52) in fresh first morning midstream urine and 80. 8% ( 42 / 52) in the fresh second morning midstream urine. While in 156 single urine samples, the positive percentages were 56.4%(88/156) and 60. 9% (95/156). The positive rates of the fresh first and second morning midstream urine were 69.7% (23/33) and 72.7% (24/33) respectively in grade 1- 2 patients, and 44.4 % (44/99) and 48. 5 % (48/99) in 99 single urine samples. The positive rates of 42 non-muscle invasive bladder cancer patients were 73. 8% (31/42) and 76.2% (32/42) in the fresh first and second morning midstream urine, while in 126 single urine samples, the positive rates were 54.8% (69/126)and 57.1% (72/126). There were no significant differences between positive rate of the fresh first and second morning midstream urine in diagnosis of bladder cancer, low grade bladder cancer and nonmuscle invasive bladder cancer. Conclusion The fresh first morning midstream urine can be used for urine cytological study in the diagnosis of bladder cancer, even in the diagnosis of low stage and low grade bladder cancer.