Objective To explore the discrimination efficiency of SDS-NaOH solution for gram-positive and gram-negative bacteria in urine. Methods Seventy nine urine samples were treated with SDS-NaOH solution for 5 minute. The bacteria accounts before and after treatment were determined by UF-1000i urine analyzer and bacteria decreased rate (BDR) was calculated. Receiver operation characteristics (ROC) curve analysis was used to assess the discrimination efficiency of BDR. Results BDR was decreased in gram-negative bacteria contained urine (P<0.01). The area under curve (AUC) of BDR for discriminating gram-positive and gram-negative bacteria was 0.70 (95% confidence interval (CI): 0.57-0.82). At cuf-off value of 0.05, the discrimination sensitivity and specificity of BDR for gram-positive and gram-negative bacteria were 0.83 (95% CI: 0.63-0.95) and 0.46 (95% CI: 0.32-0.59), respectively. Conclusion SDS-NaOH solution is useful to discriminate gram-positive and gram-negative bacteria in urine.