In this study, two novel xanthine oxidase inhibitory peptides, PDEAVAYG (820.3602 Da) and IAAGLQNTG (843.4450 Da), were purified and identified from tea protein hydrolysate, and their IC50 values were 0.09 mg/mL (109.71 μM) and 0.24 mg/mL (284.55 μM), respectively. During the gastrointestinal simulation digestion, PDEAVAYG was broken down into new peptides, while IAAGLQNTG exhibited some stability. Molecular docking results showed that hydrogen bonding, π-π stacking, and hydrophobic interactions exerted crucial effects on the interaction between peptides and xanthine oxidase. In the hyperuricemia cell model, compared to the model group, 1.0 mg/mL of PDEAVAYG and IAAGLQNTG decreased cellular uric acid levels by 40.80% and 33.33%, respectively. The RNA-seq experiments revealed that PDEAVAYG could alleviate hyperuricemia by regulating mRNA expression for pro-inflammatory factors, growth factors associated with cardiovascular disease, and uric acid efflux transporter proteins in cells. This study provides a new theoretical reference for the development of functional foods or nutritional supplements using peptides with anti-hyperuricemic activity.