Abstract: Lactate is the final metabolite of glycolysis, which is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. As an important marker in glycolysis pathway, lactate is closely related to the occurrence of cancer, diabetes, inflammation and neurodegeneration. The typical methods of chromatography and electrochemistry for lactate determination are time-consuming and complicated to operate, so the determination of lactate is usually based on the enzymatic reaction between lactate and cofactor oxidized nicotinamide adenine dinucleotide (NAD⁺). However, due to the weak fluorescence of reduced nicotinamide adenine dinucleotide (NADH) and interference from other autofluorescent biological molecules, the sensitivity of this method is low. Therefore, it’s necessary to develop a new method with improved sensitivity for lactate determination. In this paper, lactate dehydrogenase combined with genetically encoded NAD⁺/NADH sensor SoNar for determination of lactate is reported. Lactate dehydrogenase catalyzes the interconversion of lactate and pyruvate coupled with interconversion of NAD⁺ and NADH. Changes in NAD⁺, NADH, and their ratio can be sensitively and immediately recognized by SoNar. The fluorescence signal of SoNar was monitored by a microplate reader. Fluorometric detection was carried out with excitation wavelength at (420 ± 10)nm or (485 ± 10)nm and emission wavelength of (528 ± 20)nm. The detection conditions, including pH, temperature, substrate and SoNar concentration, were optimized. The lower detection limit (LOD) of this method was 2.60 μmol/L and the limit of quantitation (LOQ) was 8.67 μmol/L. The relative standard deviation (RSD) was 2.37% and the Z-factor was 0.82 in the range between 0 and 1 000 μmol/L, indicating the good stability and repeatability of this method. In addition, compared with method based on MTT-PMS, the developed novel method showed improved sensitivity, specificity and biocompatibility; and the efficiency of this method was immune to the presence of nicotinamide adenine dinucleotide phosphate (NADPH), thereby providing a useful tool for lactate metabolism studies.