As a crucial climate condition, the inter-seasonal rainfall cycle directly impacts the livelihoods and productivity of billions residing in monsoon regions. However, there is still limited understanding regarding its effects on trade-offs among ecosystem services (ES). This knowledge gap may impede the sustainable development of human well-being in these regions. In this study focused on Tana River Basin, a representative monsoon region, we assessed the seasonal supply and trade-offs of three ESs including water yield (WY), soil conservation (SC), and net primary production (NPP), which are closely linked to local human well-being. To comprehend the driving mechanism of inter-seasonal variation, the influential factors were identified using the Geographical Detector Model. Furthermore, by quantifying the seasonal trade-off intensity among three ESs, the managemental optimization priority areas were identified.The results showed that (1) all three ES supply presented an increasing trend from 2000-2020. Seasonally, both the supply and trade-offs of ESs were significantly enhanced in the rainy seasons, with spatial heterogeneity observed in the response to inter-seasonal rainfall cycle. (2) Natural condition factors presented stronger influence on the seasonal variation of ES trade-offs, which is crucial to consider the comprehensive hydrological response resulting from vegetation growth as the foundation for these seasonal variations. For the WY-SC trade-off, precipitation was identified as the primary influential factor, while vegetation coverage impact on SC-NPP and WY-NPP trade-offs. Higher precipitation and vegetation coverage would intensify the trade-off between corresponding ESs. (3) Regarding the spatial distribution of seasonal instability, several sub-watersheds aggregated in the midstream hill region were identified as the primary optimized zones, more flexible management strategies should be implemented preferentially. The findings contribute to a better understanding of how the inter-seasonal rainfall cycle impact on the ES trade-offs, supporting effective ecosystem management in monsoon regions.