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• β-Glucosidase, cellulose and phenol oxidase identified as key soil quality indicators. • Maximum tillering is an optimum growth stage of wheat for soil sampling. • Wheat yield increased significantly in puddled transplanted rice plus green manure and zero tillage with residue retention compared to residue removal. Soil quality degradation in the last four decades is threatening the sustainability of puddled transplanted rice (Oryza sativa L.) – wheat (Triticum aestivum L.) (RW) system in north-western region of India. Hence comprehensive information on the influence of soil and crop management practices in intensive and nutrient exhausting RW system on soil quality is urgently needed. The present study evaluated the effects of crop residues, green manure and tillage on the changes in soil biochemical properties at different growth stages of wheat after five cycles of RW system. Four main plot treatments in rice included combinations of wheat straw and Sesbania green manure management: (1) PTR WS0, puddled transplanted rice (PTR) with no wheat straw (2) PTR WS25 , puddled transplanted rice with 25% anchored wheat stubbles retained (3) PTR WS0 plus green manure (GM), and (4) PTR WS25 plus GM. Three sub-plots treatments in subsequent wheat included (1) CTW RS0 , conventional tillage wheat without rice straw (2) ZTW RS0 , zero tillage wheat without rice straw and (3) ZTW RS100 , ZTW with 100% rice straw retained as surface mulch. The results showed that PTR WS25 plus GM and ZTW RS100 increased significantly all the soil biochemical properties (enzyme activities and glomalin related soil protein concentration) except phenol oxidase and peroxidase activities. The activities of dehydrogenase, β-glucosidase and concentration of easily extractable glomalin and total carbohydrate carbon under ZTW RS100 were 36.8, 24.6, 25.9 and 23.3% higher than CTW RS0. Application of GM and wheat straw retention in previous rice significantly increased grain yield of subsequent wheat crop by 26.5%. The majority of the increases in biochemical properties were higher at vegetative growth (at 40–45 days after sowing) and flowering (at 80–85 days after sowing) stages compared to the initial and at maturity. Principal component analysis identified β-glucosidase, cellulase and phenol oxidase activities as the most sensitive and reliable indicators for assessing soil quality for conservation agriculture based RW system. [ABSTRACT FROM AUTHOR]