Umami peptides were screened and identified from the enzymatic hydrolysate of Spanish mackerel head and its Maillard reaction products using ultrafiltration, gel chromatography, and LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry). The umami properties of these peptides were subsequently evaluated and characterized using electronic tongue analysis and molecular docking. This study is the first to employ enzymatic hydrolysis combined with Maillard reaction for the preparation of umami peptides from Spanish mackerel head. Following this approach, a total of nine novel umami peptides were identified, including five from enzymatic hydrolysate (YDDKIY, ITPDEKGTTF, DAITTDDAGK, LEDGYPKEIQE, DAITPDEKGTTF) and four from Maillard reaction products (KDEGSDV, TPDEKGT, TEKAKGEP, FDAITPDEKGTTF). Sensory evaluation and electronic tongue analysis confirmed their distinct umami properties, with taste recognition thresholds ranging from 0.125 to 0.25 mg/mL. Molecular docking analysis revealed that these peptides interact with the T1R1/T1R3 umami receptor through hydrogen bonding and hydrophobic interactions, with key binding residues identified as Ser150, Ser256, and Glu128. This study provides a novel methodology for screening umami peptides from seafood by-products and lays the groundwork for their application as natural umami enhancers in the food industry.