Local anesthetics (LAs) are drugs that promote the reversible blocking of neural transmission by inhibiting the excitation conduction process in peripheral nerves. Tetracaine (TTC) is one of the most common topical anesthetics used in general practice and was applied to provide long-term anesthesia. In this research, poly(L-lactide) nanoparticles (PLA NPs), solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs) were utilized to construct TTC loaded nanosystems. The mean sizes, drug loading efficiency, cytotoxicity, skin permeation ability, and anesthetic analgesic effect were evaluated and compared in vitro and in vivo. The average particle sizes of blank PLA NPs, SLNs, and NLCs were 93.2, 100.9 and 110.4 nm, respectively. At all the concentrations, PLA NPs, SLNs, and NLCs showed a moderate effect on cell viability. TTC NLCs exhibited the most prominent in vivo efficiency in improving the skin permeation, analgesic time and pain control intensity. Other experiments proved that TTC PLA NPs showed advantages in serum stability and TTC SLNs illustrated the best in vitro permeation efficiency. These three kinds of nano-systems had their own superiority in some respects. Conclusion could be made that in this study, TTC NLCs is the promising system for the long-term anesthesia.