Sustainable agricultural management practices have attracted increasing attention due to their significant roles in benefiting the functions and sustainability of agro-ecosystems. An integrated agricultural practice (IP) in a maize cropping system was developed by changing row spacing, adopting no-tillage and residue return in the Northeast China. A 12-year field study was carried out to evaluate the effect of IP and conventional practice (CP) on soil physical properties, microbial biomass and enzyme activity during the cropping season. The results showed that soil organic matter under IP was increased by 17.4, 9.88 and 6.69% in June, August and October, respectively, than CP. IP enhanced microbial biomass C (by 31.7, 25.1 and 30.4% in June, August and October) and activities of invertase, urease and phosphatase (by 27.2–38.0, 78.9–182 and 9.8–29.0%) compared to CP, possibly attributing to an increase in the soil microbial community. Furthermore, the soil pH, water content, nitrogen and phosphorus contents, microbial biomass and some specific enzyme activities varied with sampling time. It is concluded that IP improved soil quality and health by increasing organic matter content and microbial biomass and activity in maize field in Northeast China, suggesting that IP is a feasible management technology for sustainable agriculture.