Abstract: In this study, soft tissues of fresh oyster (Crassostrea ariakensis) were used as the experimental material for the determination of glycogen, and the optimal reaction conditions were determined by analyzing the absorbance values obtained with different anthrone sulfuric acid ratios and at different reaction times. Then the lowest detection limit, stability and accuracy of this method were evaluated, and a trace detection system for oyster glycogen was established. The established micro-reaction system had a volume of 300 μL including 200 μL of 0.2% anthrone sulfuric acid solution and 100 μL of sample solution, and the reaction time in boiling water bath was 10 min. The minimum detection limit of glucose was 0.001 5 mg·mL⁻¹ and the coefficient of variation of the standard curve was less than 4%, indicating the high detection sensitivity and good repeatability of the method. In addition, after the reaction completed, absorbance basically remained unchanged within 120 min at room temperature, proving the high stability of this reaction. The recovery rate of glucose from six tissues including oyster mantle, gill, lip, gonad, hepatopancreas and adductor muscle were between 95.3% and 105.8%, indicating that this method has high accuracy. Thus, the established method for the determination of oyster glycogen has high repeatability, stability and accuracy, having the advantages of low reagent consumption, simple operation and low cost per sample. It is suitable for batch determination of glycogen in a large amount of samples. This study provides an effective technical method for quickly and efficiently determining glycogen in oyster samples.