Lactate concentration in blood is utilized as an indicator of tissue oxygen metabolism for assessing the therapeutic effectiveness in the treatment of sepsis. A biosensor capable of non-invasively measuring lactate produced from metabolism is in demand. The ratio of sweat lactate to blood lactate correlates strongly with the lactate threshold, the use of sweat lactate is expected to provide a non-invasive measurement. In this study, a mobile imaging system and a paper-based biosensor were developed for real-time monitoring of lactate levels in living organisms. First, a paraffin-processed channel was created on the chromatography paper using a stamp. Next, we introduce a mobile device that combines UV-LEDs and bandpass filters for high stability. We constructed an imaging system that can quantify NADH concentrations ranging from 50 μmol/L to 1 mmol/L Finally, lactate dehydrogenase (LDH) and NAD+ were dropped into the paper stream for fluorescence imaging of lactate and quantitative characterization of lactate. Fluorescence of NADH generated by LDH was confirmed, suggesting the possibility of quantifying lactate.