Cancer nanotherapeutics are rapidly progressing and being implemented to solve several limitations of conventional drug delivery systems. In this paper, we report a novel strategy of preparing methotrexate (MTX) nanoparticles based on chitosan (CS) and methoxypoly(ethylene glycol) (mPEG) used as nanocarriers to enhance their targeting and prolong blood circulation. MTX and mPEG-conjugated CS nanoparticles (NPs) were prepared and evaluated for their targeting efficiency and toxicity in vitro and in vivo. The MTX-mPEG-CS NP size determined by dynamic light scattering was 213 ± 2.0 nm with a narrow particle size distribution, and its loading content (LC %) and encapsulation efficiency (EE) were 44.19 ± 0.64% and 87.65 ± 0.79%, respectively. In vitro release behavior of MTX was investigated. In vivo optical imaging in mice proved that MTX was released from particles subsequently and targeted to tumor tissue, showing significantly prolonged retention and specific selectivity. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay obviously indicated that the higher inhibition efficiency of MTX-mPEG-CS NPs meant that much more MTX was transferred into the tumor cells. A significant right-shift in the flow cytometry (FCM) assay demonstrated that MTX-loaded nanoparticles were far superior to a pure drug in the inhibition of growth and proliferation of Hela cells. These results suggest that MTX-mPEG-CS NPs could be a promising targeting anticancer chemotherapeutic agent, especially for cervical carcinoma.