The tracking，monitoring and evaluation of flooding performance in microbial enhanced oil recovery process was particularly important，so rapid quantitative analysis of biological surfactant became an important technical support. In order to rapid quantitative determination of rhamnolipid with low concentration，standard curve of rhamnolipid was obtained by optimized phenol-sulfuric acid method，oil spreading method and high performance liquid chromatography-electrospray mass spectrometry（HPLC-MS）. The results showed that the optimum acidification condition of phenol-sulfuric acid method was obtained as follows: 1 h acidification time and 80℃ acidification temperature. The phenol-sulfuric acid method could quickly quantitative measure 100—1000 mg/L rhamnolipid. The relationship of absorbance（A）and rhamnolipid concentration（c）was A=7.465×10^-4 c+0.047，with 0.9939 R²，showing good linear relationship. Rhamnolipid with concentration of 20—500 mg/L could be quickly approximate quantification by oil spreading method. The R² of rhamnolipid concentration and oil zone diameter standard curve was less than 0.95，showing large error. Rhamnolipid with concentration of 1—150 mg/L could be determined accurately by HPLC-MS. The relationship between the absorption peak area S and c was S=3085.4c-2201.4，with 0.9993 R²，showing good linear relationship. Phenol-sulfuric acid method，oil spreading method could be used to fast quantitative analysis of rhamnolipid on site，while HPLC-MS could be used to accurate quantitative analysis indoors.