In this work, an acid invertase isolated from Lathyrus aphaca seedlings is purified, and its thermal properties are investigated. Acid invertase was purified through salt fractionation, CM-cellulose, and Ultrogel ACA-44 chromatography. Using SDS-PAGE, the consistency of the isolated enzyme was verified. The enzyme weighs 29 kDa at the molecular level. Acid invertase functions best at a pH of 3.0 and a temperature of 45 °C. Activation energy of 29.5 kJ mol−1 and Km and Vmax values of 0.5 mM and 119.7 µmol. min−1. mg−1 of protein was found for the isolated enzyme. It was shown that the mercaptide-forming agent p-chloromercuribenzoic acid (PCMB; 0.5 mM) and the enzyme's activity were both modestly increased by Ca2+, Mn2+, and Mg2+ ions but inhibited by Hg2+, Cd2+, and Pb2+ ions. At elevated temperatures, the invertase exhibited an increase in thermostability due to an increase in activation entropies (∆Sº) and a decrease in activation enthalpies (∆Hº). The interactions between 4 M urea and α-chymotrypsin were tetraphasic, causing periodic increases and decreases in invertase activity. It also suggests a potential explanation for acid-invertase thermal inactivation at high temperatures. This enzyme might be a catabolite-resistant invertase to produce high-gravity ethanol or fructose syrup.