Systemic study on thermal dilute-acid hydrolysis of cotton straw has been conducted. The process of sequential two-step reaction has been established, which can greatly improve the utilization efficiency of lignocellulosic biomass. The hydrolyses were carried out in an 1L erlenmeyer flask placed in the pressure steam sterilizer, where the hydrolysis retention time (0-60min), liquid to solid ratio (8-14:1) and acid concentration (0-1mol/L) were examined for the first stage. Optimization of first stage hydrolysis is desirable to achieve the highest yield of the sugars from hemicellulose and also as a pretreatment for the second acid hydrolysis. The solid residuals were further dilute-acid (0.2mol/L) hydrolyzed in this study. This second-stage hydrolysis mainly completed the conversion of cellulose to levulinic acid and formic acid. At last, dilute-acid conversion of cotton straw to sugars (in the first stage), levulinic acid and formic acid (in the second stage) was completed.