Understanding pesticide behavior in plants is important for effectively applying pesticides and in reducing pesticide exposures from the ingestion. This study aimed to investigate the penetration and persistence of pesticides applied on harvested and live basil leaves. Surface-enhanced Raman scattering (SERS) mapping was applied for in situ and real-time tracking of pesticides over time using gold nanoparticles as probes. The results showed that after surface exposure of 30 min to 48 h, pesticides (10 mg/L) penetrated more rapidly and deeply into the live leaves than the harvested leaves. Systemic pesticide thiabendazole and the non-systemic pesticide ferbam can penetrate into the live leaves with depth of 225 μm and 130 μm, respectively than the harvested leaves with depth of 180 μm and 18 μm, respectively after 48-h exposure. The effects of leaf integrity and age on thiabendazole penetration were also evaluated on live basil leaves after 24-h exposure. Thiabendazole (10 mg/L) when applied onto intact leaves penetrated deeper (170 μm) than when applied onto damaged leaves (80 μm) prepared with 20 scrapes on the top surface of leaves. Older leaves with a wet mass of 0.204 ± 0.019 g per leaf (45 days after leaf out) allowed more rapid and deeper penetration of pesticides (depth of 165 μm) than when younger leaves with a wet mass of 0.053 ± 0.007 g per leaf (15 days after leaf out) were used (depth of 95 μm). The degradation of thiabendazole on live leaves was detected after 1 week whereas the apparent degradation of ferbam was detected after 2 weeks. In addition, the removal of pesticides from basil was more efficient when compared with other fresh produce possibly due to the specific gland structure of basil leaves. The information obtained here provides a better understanding of the behavior and biological fate of pesticides on plants.