Stimuli-responsive materials, enabling drugs to be released in the acidic tumor and intracellular microenvironments, draw an increasing attention in chemotherapy. Here novel pH-sensitive biodegradable micelles are fabricated using a one-step, one-medium process without organic solvent for efficient loading and rapid intracellular release of hydrophobic cargos. The amphiphilic chondroitin sulfate-histamine conjugate (CS-his) were successfully synthesized and assembled into nanoparticles in aqueous medium with desirable size (133 nm) and low critical micelle concentration (CMC) (0.05 mg/L). Owning to the pH-sensitive structure of imidazole, the nanoparticles show pH-responsive behavior upon reducing the pH value of surrounding media, accompany with formation of large aggregates and increase of ζ potential. When the nanoparticles were utilized to deliver the model drug DOX, they exhibited a specific on-off switch drug release behavior, triggering DOX release in acidic surroundings (intracellular endosomes) and sealing DOX in neutral surroundings (blood circulation or extracellular matrix). CCK-8 assays and confocal laser scanning microscopy (CLSM) against HepG2 cells indicated that the nanoparticles themselves had no associated cytotoxicity, while drug-loaded nanoparticles possessed high cytotoxicity to model cells and presented high efficiency of cellular uptake. These flexible micelles with an on-off switched drug release may offer a promising pattern to accurately deliver a wide variety of hydrophobic payloads to tumor cells for cancer therapy.