DL-Lactic acid and D-glucose are important human health indicators. Their aberrant levels in body fluids may indicate a variety of human pathological conditions, suggesting an urgent need of daily monitoring. However, simultaneous and rapid analysis of DL-lactic acid and D-glucose using a sole but simple sensing system has never been reported. Here, an engineered Mycobacterium smegmatis porin A (MspA) nanopore is used to simultaneously identify DL-lactic acid and D-glucose. Highly distinguishable nanopore event features are reported. Assisted with a custom machine learning algorithm, direct identification of DL-lactic acid and D-glucose is performed with human serum, demonstrating its sensing reliability against complex and heterogeneous samples. This sensing strategy is further applied in the analysis of different animal serum samples, according to which gluconic acid is further identified. The serum samples from different animals report distinguishable levels of DL-lactic acid, D-glucose and gluconic acid, suggesting its potential applications in agricultural science and breeding industry. This sensing strategy is generally direct, rapid, economic and requires only ≈µL of input serum, suitable for point of care testing (POCT) applications.