The complexity, degradability, and stability of drug delivery systems are crucial factors for clinical application. Herein, a glutathione (GSH)-responsive polyethylene glycol (PEG)ylated nanogel conjugated with doxorubicin (Dox) was prepared based on a linker with disulfide bonds, PEG, and Dox using a one-pot method. FT-IR and UV-vis analyses confirmed that all raw materials were incorporated in the Dox-conjugated nanogel structure. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) results showed that the particle size of the Dox-conjugated nanogel was at the nanoscale and could be responsively disrupted in high GSH concentration. The in vitro accumulative Dox release rate from the nanogel reached 88% in PBS with 5 mg mL-1 GSH on day 4. Moreover, H22 cell viability and apoptosis experiments revealed that the nanogel effectively inhibited tumor cell growth. In vivo tracking and cell uptake experiments demonstrated that the nanogel accumulated and persisted in tumor tissues for 5 days and was distributed into cell nuclei at 6 h. Furthermore, H22-bearing mice experiments showed that the tumor size of the Dox-conjugated nanogel group was the smallest (287 mm3) compared to that of the free Dox (558 mm3) and 0.9% NaCl (2700 mm3) groups. Meanwhile, the body weight of mice as well as the H&E and TUNEL tissue section staining of organs and tumor tissues from the mice illustrated that the nanogel could significantly prevent side effects and induce tumor cell apoptosis. Taken together, compared with free Dox, the Dox-conjugated nanogel exhibited higher therapeutic efficacy and lower side effects in normal tissues, making it a potential novel nanomedicine for cancer.