Tomato wilt is caused by Fusarium oxysporum f. sp. lycopersici (FOL), a soilborne pathogen causing global economic losses. FOL aggressive isolates produce tomatinase to counteract tomato defense. The study aimed to identify and analyze the tomatinase gene in three FOL isolates with varying aggressiveness. Genetic analysis was employed using a polymerase chain reaction-based method, and the sequences were compared with several tomatinase sequences. Post-translational functional analysis predicted phosphorylation sites for the three tomatinase sequences using NetPhos3.1 server. Results showed that FO-19, the most aggressive isolate, had the highest tomatinase activity. Tomatinase activity was found to be correlated with phosphorylation, which may be quite important in the aggression of FOL isolates. The study highlights the importance of understanding the FOL-tomato interaction in developing effective strategies to control tomato vascular wilt.