The novel amphiphilic derivatives of Methotrexate-chitosan oligosaccharide (MTX-CHO) with different molar feeding ratios of MTX were synthesized. The degree of MTX substitution ranged from 4.47 to 13.5%. MTX-CHO copolymer formed micelles with an average size of 134.6±14.52 to 236.6±30.01 nm, and zeta potential of 20±5 to 16.8±7.74 mV. The critical micelle concentration was found to range from 125 to 0.56 mg/l. Analysis of micelles with different degree of substitutions (DSs) revealed that the size of micelles decreased by increasing DS while zeta potential was reduced. Release study indicated that drug content had effect on the release rate. With increasing amount of loaded drug in the micelle, release rate was decreased. Drug loaded and unloaded MTX-CHO micelles showed significant cytotoxicity on MDA-MB-231. Loaded micelle was more effective than unloaded one which indicated that conjugation could reduce efficacy of MTX. The viability of MDA-MB-231 in presence of drug loaded micelles was significantly decreased and cell viability at 1 µg/ml was 45.17±9% while the viability of unloaded micelles was 91.86±9.88. These phenomena make MTX-CHO micelles as a good candidate for hydrophobic anticancer drug carrier.