Heterogeneous catalysis over single-atom catalysts (SACs) has garnered significant attention in biomass-derived platform chemical conversion owing to the high atomic utilization and reliable selectivity/stability. Herein, Co single-atom catalysts (Co–N/F1) derived from fractionated lignin were employed for the oriented oxidation of 5-hydroxymethylfurfural (HMF), a typical platform chemical derived from glucose, into 2,5-furandicarboxylicacid (FDCA) under mild conditions. The Co–N/F1 with enhanced Co content was obtained using the low-molecular-weight lignin fraction (F1) with high functional group contents (e.g., Ph–OH and COOH), and the as-prepared catalyst was demonstrated to present a Co–N4 configuration. Owing to the absence of colored byproduct formation from HMF at elevated temperatures, Co–N/F1 realized the highly selective conversion of HMF to FDCA (100% HMF conversion, 99.20% FDCA yield) under mild conditions (65 °C, 3 bar O2), which outperformed most reported non-noble metal-supported catalysts. Density functional theory calculations indicate that the Co–N4 site in Co–N/F1 facilitates the dehydrogenation of the α-C position on HMF and its aldehyde intermediates, resulting in a significantly enhanced catalytic efficiency. Furthermore, Co–N/F1 exhibited stable reusability due to the alkaline resistance of the Co–N4 structure. Our study details the insights into the synthesis of Co-SACs using a lignin fractionation strategy and further demonstrates its good feasibility for the oriented conversion of biomass-derived platform chemicals under mild conditions.