PurposeMore information is needed on the comparative evaluation of C sequestration and soil fertility conservation between straw and biochar application. Particularly, the effect of the co-application of straw and biochar on the quantity and quality of soil organic matter (SOM) remains unknown.MethodsA 3-year field experiment was conducted in a Mollisol, and included the following treatments: no application (CK), straw (CS) and biochar application (BC), and mixing straw with biochar (BS). C groups were identified by solid-state C-13 NMR.ResultsApplying straw and biochar significantly increased the contents of SOM (10.02-20.99%), labile and humic C, and corn yield (11.01-22.78%), compared to CK. BC had a more significant potential to increase SOM, C pool index, and corn yield than CS, while CS had the highest C lability index and C management index. BC improved the hydrophobicity and aromaticity, whereas CS increased the aliphatic and hydrophilic properties in SOM chemical composition. BS greatly reduced the ratios of alkyl C/O-alkyl C and aliphatic C/aromatic C.ConclusionsBC is more conducive to the stability and accumulation of SOM, while CS is beneficial to exerting soil fertility attributed to the improvement in the bioavailability and reactivity of SOM. BS could synergistically improve the biological activity and stability of SOM to some degree. Considering biochar production cost, we recommend introducing a small amount of biochar into straw returning practice as a booster to promote the stability and activity of SOM and consequent crop production, synergistically realizing a low C strategy and fertility conservation.