In this work, we present a lactate-selective potentiometric sensor as simple, low cost and non-enzymatic alternative for application in three different biological fluids. The selectivity of proposed sensor is based on a polypyrrole matrix doped with lactate ions in its structure which occurs during galvanostatic polymerization step. After optimization, the device showed a wide linear range between 0.1 and 10.0 mmol L−1 with a slope of 23.7 ± 0.2 mV dec−1, a limit of detection (LOD) of 81 μmol L−1, and excellent repeatability (RSD = 2.1%) and reproducibility (RSD = 2.7%). Tests to evaluate the influence of possible interferences were performed, in which the sensor showed good selectivity to lactate ions. The sensor was applied to evaluate lactate levels in human tear samples and a linear correlation with lactate levels in blood samples (R2 = 0.977) was found. In order to assess lactate levels before and after intense physical activity, blood samples from rats and human sweat samples were analyzed. For both fluids the potential variation was obtained, and in comparison to the lactate analysis in blood by portable device, the human sweat response was confirmed by the proposed method. Thus, the developed sensor demonstrated a viable and efficient electroanalytical tool to the current commercial devices, especially to support in physical performance evaluations.