Context Biological quality of soil under rice-based cropping in Indo-Gangetic Plains of India is greatly influenced by different tillage and residue management practices. Aims The objectives of this study were to determine the effect of seasonal variation on enzyme activity and assessing biological soil quality (BSQ) under conservation agriculture (CA)-based rice–mustard cropping. Methods Soil samples were collected in the ninth year of a medium-term CA-based experiment in autumn, winter, spring and summer seasons from two soil depths (0–5 cm, 5–15 cm) and analysed for biological attributes (microbial biomass carbon, MBC; Walkley–Black carbon, WBC; total organic carbon, TOC), and enzyme activities (dehydrogenase, acid and alkaline phosphatase, and arylsulphatase). Key results A CA-based triple zero till (ZT) system with three crops residue treatments (ZT direct-seeded rice (DSR) with mungbean residue; ZT mustard (ZTM) with rice residue; and ZT summer mungbean (SMB) with mustard residue (∼ZTDSR–ZTM–ZTSMB (+R)) resulted in highest values of MBC, WBC and TOC at the 0–5 cm soil depth. Enzymes activities were highest during summer. Principal component analysis (PCA) and showed that arylsulphatase activity and TOC were sensitive indicators of BSQ. To validate soil quality indices (SQIs), regression analyses with rice equivalent yield (REY) of the rice–mustard system were done and better relationship observed with biological soil quality indices (BSQI) BSQIGMean (0.69–0.71) than BSQIPCA (0.65–0.66) based method. Conclusions Treatment with ZTDSR–ZTM–ZTSMB (+R) had the highest values of BSQI using GMean and PCA. Implications This CA-based triple ZT with residue practice [ZTDSR–ZTM–ZTSMB (+R)] is recommended for rice–mustard cropping for maintaining higher biological quality of soil in this region of India.