This study aims to investigate the feasibility of measuring the polysaccharide content by a full-wavelength microplate reader instead of the ultraviolet spectrophotometer, and to verify the reliability of the results. First, the glucose content of the glucose standard solution was detected by ultraviolet spectrophotometer and full-wavelength microplate reader, respectively, to confirm the feasibility of measuring the polysaccharide content by a full-wavelength microplate reader instead of the ultraviolet spectrophotometer. Then, the reliability of the full-wavelength microplate reader for detecting polysaccharide content was investigated and the measurement conditions were optimized. Finally, using this method to detect the polysaccharide content of natural plant polysaccharide compound. The results showed that the full-wavelength microplate reader could replace the ultraviolet spectrophotometer to determine the polysaccharide content. For full-wavelength microplate reader method, the relative standard deviation (RSD) value of precision was 0.41%, the RSD value of sample repeatability was 1.06%, the RSD value of standard repeatability was 0.84%, the recovery rate was 100.02%, the RSD value of sample stability was 1.25%, and the RSD value of standard stability was 1.34%. The main influencing factors including dilution multiple, detection wavelength, sulfuric acid addition and water bath temperature were screened. The determination conditions of polysaccharide content in compound polysaccharides of natural plant polysaccharide compound by full-wavelength microplate reader-phenol-sulfuric acid method were as follows:sample was treated and diluted 200 times into a testing solution, determined as 5 mL of sulfuric acid, 30℃ water bath temperature, and 490 nm detection wavelength. Through measuring, the polysaccharide content in the natural plant polysaccharide compound is 114.70 mg/g.