Optimization of conditions for quantitative determination of the content of poly(β-malic acid)in fermentation broth was established by RP-HPLC.Methods:A(0.01 mol/L KH2PO4)∶B(methyl cyanide)= 95∶5 as mobile phase,using column Synergi 4u Polar-RP(4.6 × 250 mm,5 μm),Agilent 1200 series liquid color spectrum system to measure the contents of malic acid before and after acid hydrolysis,and their difference in value reports the content of poly(β-malic acid)in fermentation broth indirectly.Results showed that the target peaks and impure peaks were completely separated within 8 minutes,quantitative relative standard deviation is 0.6%～1.5% and the recovery is 98.2%～100 %.This method can be used to detect poly(β-malic acid)in fermentation broth with high accuracy and good reproducibility.