Stearic acid-grafted chitosan oligosaccharide (CSO-SA) with 3.48% amino-substituted degree (SD%) was synthesized by coupling reaction. The CSO-SA could self-aggregate to form micelle with a critical micelle concentration (CMC) at 0.035 mg/mL in the aqueous phase. The CSO-SA self-aggregate micelles indicated spatial structure with multi-hydrophobic core. One CSO-SA chain could form 2.8 hydrophobic cores. Cellular uptakes of CSO-SA micelles by using A549, LLC, and SKOV3 cells as model tumor cell lines showed the faster cellular internalization of CSO-SA micelles, and the cellular uptakes on the LLC and SKOV3 cells were higher than that on the A549 cells. Doxorubicin (DOX) was then used as a model drug to incorporate into CSO-SA micelles. To reduce the initial burst drug release from CSO-SA micelles loading DOX (CSO-SA/DOX), the shell of CSO-SA micelles was crosslinked by glutaraldehyde. The shell crosslinking of CSO-SA micelles reduced the micelle size and surface potential, but it did not significantly affect the cellular uptake and drug encapsulation efficiency of CSO-SA micelles. The cellular inhibition experiments demonstrated that the cytotoxicity of DOX was increased by the encapsulation of CSO-SA micelles. CSO-SA/DOX displayed the best antitumor efficacy in SKOV3 cell line due to the higher cellular uptake percentage of CSO-SA micelles and the lower sensitivity of free drug to the cells. The cytotoxicities of shell crosslinked CSO-SA/DOX were highly enhanced in all cell lines than those of unmodified CSO-SA/DOX.