Labile soil organic matter (SOM) fractions are especially important because they are more vulnerable to disturbance and play a crucial role for nutrient and carbon cycling. Although water conservation has become increasingly important in rice–wheat rotation, the effects of non-flooded mulching cultivation on labile SOM fractions remain unknown. Based on a long-term field experiment (10 years), we analyzed the impact of non-flooded mulching cultivation and cropping season on labile SOM fractions in a rice–wheat rotation in Chengdu Plain, southwest China. Compared with traditional flooding (TF), the plastic film mulching (PM) and wheat straw mulching (SM) treatments increased dissolved organic carbon (DOC) (42% after rice season and 41% after wheat season), but decreased microbial biomass carbon (MBC) (19% after rice season and 28% after wheat season) and nitrogen (MBN) (17% after rice season and 24% after wheat season) in the 0–5 cm depth. SM increased particulate organic carbon (POC) and KMnO4-oxidizable C (KMnO4-C) contents after both the rice and wheat seasons. Microbial biomass and DOC concentrations were higher for all three cultivations after the rice season than after the wheat season. In contrast, POC contents under PM and SM were higher after the wheat season than after the rice season. In general, results in this study indicate that non-flooded mulching and cropping season significantly influenced labile SOM fractions. The DOC fraction was the most sensitive fraction affected by non-flooded mulching, while POC and PON fractions respond fast within the two cropping seasons.