Detritus is the vital support for the microbial food web, which would further affect river ecological conditions. Determining the effects of detritus availability on microbial food webs in rivers is critical for protecting river ecological functions. However, the detritus availability was difficult to estimate directly, since the detritus transformation processes (i.e. detritus availability) and flow-induced transport processes are interdependent in rivers. Therefore, this study quantified the detritus transformation processes in a natural river and further identified the impacts of detritus availability on microbial food web patterns. Results revealed that the flow velocity was the main physical driver determining the detritus availability. The decreased velocity would promote detritus availability. Moreover, the increased detritus availability significantly promoted the diversity of bacteria, protozoan and metazoan (p < 0.05). The responses of low trophic levels to detritus availability were significantly greater than those of higher trophic levels, emphasizing the bottom-up cascading effect of detritus availability on microbial food web composition (p < 0.05). From microbial food web perspectives, the detritus availability was amplified with flow velocity decreased, promoting trophic transfer efficiency between different trophic levels. Results and findings revealed the ecological effect of detritus transformation processes on multi-trophic levels in rivers and provided advantageous information for river management.