Cell metabolite detection is important for cell analysis. As a cellular metabolite, lactate and its detection play an important role in disease diagnosis, drug screening and clinical therapeutics. This paper reports a microfluidic chip integrated with a backflow prevention channel for cell culture and lactate detection. It can effectively realize the upstream and downstream separation of the culture chamber and the detection zone, and prevent the pollution of cells caused by the potential backflow of reagent and buffer solutions. Due to such a separation, it is possible to analyze the lactate concentration in the flow process without contamination of cells. With the information of residence time distribution of the microchannel networks and the detected time signal in the detection chamber, it is possible to calculate the lactate concentration as a function of time using the de-convolution method. We have further demonstrated the suitability of this detection method by measuring lactate production in human umbilical vein endothelial cells (HUVEC). The microfluidic chip presented here shows good stability in metabolite quick detection and can work continuously for more than a few days. It sheds new insights into pollution-free and high-sensitivity cell metabolism detection, showing broad application prospects in cell analysis, drug screening and disease diagnosis.