In situ monitoring of the uptake and transportation process of nano-pesticides during crop growth remains challenging thus far. In this report, the different three sizes of surface-enhanced Raman spectroscopy probes of nano-pesticides loaded with ferbam and acetamiprid are representative non-systemic or systemic pesticides, respectively. The probes were verified to have strong signals of the Raman spectrum enhancement effect. They were further applied on the leaves and roots of tea plants. Within 5 h, the nano-pesticides of 50 nm and 100 nm except 150 nm probes could rapidly penetrate young tea leaves to a depth of about 150−290 μm. Whereas none of the probes were observed to penetrate mature leaves and roots of tea plants. The result showed that the penetration rate depended on the size of the nano-pesticides and the structure or maturity of the plant tissue rather than the loaded pesticides. The penetration rate of small-sized nano-pesticides in young tea leaves is the fastest.