This study addressed the issues of insufficient sensitivity and poor stability of traditional Coomassie Brilliant Blue method in trace protein detection by systematically optimizing each procedural step based on its reaction principles. Firstly, a microplate reader combined with 96-well plates was employed to replace traditional ultraviolet spectrophotometers and cuvettes, thereby improving detection throughput and precision. In addition, 20 mmol/L phosphate buffer (PB) was used as the diluent instead of water to enhance protein solubility. Lastly, the optimal formulation of Coomassie Brilliant Blue G-250 solution was prepared by dissolving 5 mg Coomassie Brilliant Blue G-250 in an aqueous solution containing 5% of 95% ethanol and 13% of 85% phosphoric acid. Validation results demonstrate that, the optimal reaction system involved an equal volume of bovine serum albumin (BSA) mixed with the Coomassie Brilliant Blue G-250 solution, with the ideal reaction conditions being a 10-minute incubation in the dark. The method demonstrates excellent linearity (R2>0.99) within the protein concentration range of 0~21 mg/L, with a detection limit as low as 1 μg/mL. Both satisfactory accuracy (80%~115%) and precision (RSD<4%) meet pharmacopoeia reference standards. This study successfully achieves accurate quantitative analysis of trace proteins, offering a cost-effective, rapid, and high-throughput approach, thereby providing more precise and efficient technical support for protein-related research.