In order to improve the adsorption capacity of chitosan (CTS) for methylene blue (MB) cationic dye, a series of N,O-carboxymethyl-chitosans (N,O-CMCTS) were prepared under heterogeneous conditions by controlling the reaction time. The adsorption of MB from aqueous solution onto N,O-CMCTS was studied. The effects of degree of substitution (DS) of N,O-CMCTS, initial pH of the dye solution and adsorption temperature were investigated in detail. The results showed that the adsorption capacities of N,O-CMCTS increased with the increase of DS and N,O-CMCTS with DS of 0.72 exhibited the highest adsorption capacity (349 mg/g). The adsorption kinetics of N,O-CMCTS was found to follow the pseudo-second-order model. The adsorption equilibrium of N,O-CMCTS fitted very well with the Langmuir isotherm model, showing maximum monolayer adsorption capacity of 351 mg/g. The adsorption mechanism of N,O-CMCTS was also discussed by means of FTIR spectra and XPS. The results revealed that the -OH, -NH2, and -COOH groups of N,O-CMCTS were involved in the adsorption process. The desorption studies showed that N,O-CMCTS could be regenerated and used for the adsorption of MB repeatedly.