Ethyl carbamate (EC) is a naturally occurring component in the fermented products, especially the Chinese yellow rice wine. EC has been found showing a potential for carcinogenity and causing food safety problems. This study is to investigate the application of the existing HPLC-FLD method to the analysis of EC in Chinese yellow rice wine, and to validate it for EC analysis with high through-out and applicability in Chinese yellow rice wine sample. The parameters examined in this study were fully evaluated. Results indicated that good linearity was obtained with a correlation coefficient exceeding 0.9990, the limit of detection and quantification was 73.2 and 243.9 μg L−1, respectively. Recoveries ranged between 98.30% and 101.30%, and the precision of this method was lower than 5% (RSD). The method conducted in this work was successfully applied to determine EC concentration in Chinese yellow rice wine samples from different origins. The negative correlation between EC formation and urea level in yellow rice wine samples is evaluated with the developed HPLC-FLD method.