Peptide self-assemble hydrogels are a group of functional biomaterials utilized as vehicles for drug delivery. The gelation of tetrapeptides, Fmoc-RFFD-NH₂, including R (arginine), two F (phenylalanine) and D (aspartic acid) amino acids, was in deionized water, PBS (phosphate buffer saline, pH 7.4) or saline. The three-dimensional (3D) network architectures of hydrogel were revealed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Puerarin, the model drug, was incorporated in the hydrogel and its secondary structure change was assessed by circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy. The interaction of puerarin and Fmoc-RFFD-NH₂ was evaluated by molecular modeling. Puerarin released from the hydrogel in pH 7.4 solution for 10 h. Additionally, the effects of the puerarin hydrogel (P-Gel) on rats with monoiodoacetic acid (MIA)-induced osteoarthritis (OA) were evaluated, and the hydrogel demonstrated excellent histocompatibility without cytotoxicity. Finally, compared to the blank hydrogel, P-Gel acted on rats and the thickness of the articular cartilage were substantially enhanced, and the loss of proteoglycan was reduced. In summary, the peptide hydrogel is an effective and safe strategy in biological applications for drug delivery to treat OA using puerarin.