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Chitosan is a natural basic polysaccharide, currently which have no a very effective way to determine its content. In this paper, we used mixed acid and ultrasound-assisted with chitosan which hydrolyzed into glucosamine, under alkaline condition acetyl acetone may react with paradimethylaminobenzaldehyde acid alcohol solution to form red compound, which was measured at 525nm by spectrophotometry. More appropriate technological conditions have been determined. The results showed that the optimal conditions for quantity of chitosan was 2mg, 0.168mol/L hydrochloric acid was 0.8ml, 75% sulfuric acid was 3.5ml, in boiling water bath for 30min, supersonic for 60min at 50 ℃, the average recovery was 96.9±3.24%, the content of chitosan was 87.89±1.02%, the production rate of chitosan was 84.79±0.99%. The results show that the mentioned method is easy and effective for the quantitative analysis of chitosan by spectrophotometry.