Conservation farming has been recognized as effective climate strategies for enhancing soil organic carbon (SOC) sequestration and soil health. Yet, the extent to which this alternative agriculture influences SOC characteristics (i.e., biomolecules, lability and sources) in the soil matrix remains elusive. Employing three biomarkers – lipids, lignin and microbial necromass – we differentiated the composition, degradation and sources of two functional forms of organic matter: particulate (POM) and mineral-associated fractions (MAOM) in response to 20-year conservation practices in North China. Three treatments included: conventional tillage (CT), rotary tillage (RT), and no-till (NT). Topsoil samples (0–5 cm) were subjected to physical and chemical fractionation. The SOC content in POM was 40.8% higher in RT and 59.2% higher in NT compared with CT, whereas the increased SOC in MAOM was limited (by 1.3% and 8.9%). NT (cf. CT) increased short-chain lipids (<C20) by 42.2% in the MAOM fraction. Compared with CT, NT increased lignin phenols by 31.8% in POM and by 33.3% in MAOM, reflecting the preservation of plant-derived compounds. Again, NT and RT augmented microbial necromass C (MNC) by 130.0% and 77.2% in POM rather than MAOM relative to CT, restoring more microbial residues in the particulate form. NT (cf. CT) also enhanced the ratios of MNC/SOC and fungal MNC/bacterial MNC in the POM rather than MAOM. We collectively concluded that conservation tillage altered SOC biochemistry and accrual pathways via restoring lignin phenols and microbial necromass in distinct fractions, highlighting a novel stabilization mechanism of plant- and microbial-derived biomolecules under alternative systems.