A set of experiments were conducted to screen the most thermo‐resistant microorganism and spoilage enzyme in sweet lime juice. Furthermore, the impact of matrix pH on the batch thermal treatment (75–95°C/0–10 min) intensities on Saccharomyces cerevisiae survival, PPO inactivation, and vitamin C retention in the sweet lime juice was evaluated. The influence of cumulative come‐up time (tCUT) and cooling time (tCOOL) was separated from the isothermal holding period (tHOLD). All three responses fitted best to the first‐order kinetics. The rate constants (k) ranged between 0.0053 and 0.471 s−1 (for S. cerevisiae), 0.00114 and 0.00731 s−1 (for PPO), and 0.000017 and 0.000142 s−1 (for vitamin C). The microbial and enzyme inactivation rate increased at a low pH level for a certain temperature, whereas, for vitamin C, an opposite trend was prominent through marginal. A new global kinetic model for k as a function of matrix pH and the temperature was developed in which the sensitivity of k to pH change was introduced as SpH. The optimized thermally pasteurized juice (95°C/298 s of holding time) at pH 3.5 showed >5 log cycle reduction in S. cerevisiae count (t5D, s) and 99% inactivation in PPO activity (t99, s) while retaining 57% vitamin C and 39% loss in total phenolics. The optimally treated pasteurized sample at pH 3.5 was examined for phenolic profiling, microbial cell morphology, and enzyme conformational change. The thermally pasteurized sample had a sensory acceptance of 6.9 out of 9 scales.Practical applicationsThe present study developed a systematic study for designing a thermal pasteurization condition for sweet lime juice. First, the most thermo‐resistant microorganism and spoilage enzymes in sweet lime juice were screened out based on the sensitivity index. Further, a global kinetic model was developed for the most resistant microorganisms, resistant enzymes, and vitamin C. This model can describe the impact of matrix pH and temperature on the treatment time to achieve microbial safety and enzymatic stability. Pasteurization time was estimated for each combination of pH and temperature. Assessing the impact of pH levels in the matrix on the effectiveness of thermal pasteurization would assist the industry in determining optimal harvesting conditions for fruit with the desired pH.