Nanoparticles that have been developed to effectively deliver therapeutic drugs and imaging agents to tumor regions could increase therapeutic efficacy while dramatically reducing potential side effects with the capability of diagnosing tumors. In this review article, we discuss the development and optimization processes of glycol chitosan-based polymeric nanoparticles to accomplish the tasks of therapeutic drugs and imaging agents for cancer treatment. To achieve effective tumor imaging and therapy, we optimized the physicochemical properties of nanoparticles and examined several key factors (i.e., molecular weight, stability in the blood, deformability, and rapid cellular uptake), which are important issues influencing the in vivo biodistribution and tumor targeting ability of nanoparticles. Furthermore, we showed their applications in various animal tumor imaging and therapy studies. This review article suggests that the optimization processes for developing novel nanoparticle systems are necessarily required to obtain the most effective therapeutic drugs and imaging agents through in vitro and in vivo studies.