The aim of this investigation is preparation of Mitomycin-C encapsulated with chitosan nanoparticles synthesis using ionic gelation technique for intravesical controlled drug delivery systems. This study was conducted in vitro. Cumulative amount of drug released from the nanoparticles was calculated. Mitomycin-C release studies were examined for different pH values. During the drug loading and release studies, initial amount of drug was changed (i.e., 0.5, 1.25 and 2.5 mg) to get different release profiles and the release studies were repeated (n = 6). The loading efficiencies of Mitomycin-C with three different initial concentrations 0.5mg/ml, 1.25 mg/ml and 2.5 mg/ml into chitosan nanoparticles were 54.5%, 47.1% and 36.4%, respectively. For different pH values, the cumulative releases of Mitomycin-C from chitosan nanoparticles were 47% and 53% for pH 6.0 and 7.4, respectively (p < 0.01). For different drug doses, the cumulative releases of Mitomycin-C (MMC) from Chitosan nanoparticles were 44%, 53% and 65% for 0.5 mg/mL, 1.25 mg/mL and 2.5 mg/mL respectively (p < 0.01). The anticancer activity of Mitomycin-C loaded chitosan nanoparticles was measured in T24 bladder cancer cell line in vitro, and the results revealed that the 2.5 MMC coated Chitosan nanoparticles had better tumor cells decline activity. From this investigation, we conclude that the drug encapsulated synthesized chitosan nanoparticles possess a high ability to be used as pH and dose responsive drug delivery system. This systematic investigation demonstrates a promising future for the intravesical installation in treatment of the superficial bladder cancer.