Knowing the concentrations of biological substances can help ascertain physiological and pathological states. In the present study, a minimally invasive microperfusion needle was developed for measuring the concentrations of biological substances in subepidermal tissue. The microperfusion needle has a flow channel with a perforated membrane through which biological substances from subepidermal tissue are extracted. Since this device uses a thin steel acupuncture needle as the base substrate, it has sufficient rigidity for insertion through the skin. The efficacy of the needle was examined by measuring lactate and glucose concentrations in mice. Lactate was injected intraperitoneally, and changes in lactate concentrations in subepidermal tissue over time were measured using the device. Lactate concentrations of blood were also measured as a reference. Lactate was successfully collected using the microperfusion needle, and the lactate concentration of perfused saline was significantly correlated with blood lactate concentration. Glucose solution was administered orally, and the glucose concentration of perfused saline was also correlated with blood glucose concentration. The newly developed microperfusion needle can be used for minimally invasive monitoring of the concentrations of biological substances.