Whiteleg shrimp (Penaeus vannamei) have been vulnerable to the stress induced by different aquaculture operations such as capture, handling, and transportation. In this study, we developed a novel clove oil-nanostructured lipid carrier (CO-NLC) to enhance the water-soluble capability and improve its anesthetic potential in whiteleg shrimp. The physicochemical characteristics, stability, and drug release capacity were assessed in vitro. The anesthetic effect and biodistribution were fully investigated in the shrimp body as well as the acute multiple-dose toxicity study. The average particle size, polydispersity index, and zeta potential value of the CO-NLCs were 175 nm, 0.12, and −48.37 mV, respectively, with a spherical shape that was stable for up to 3 months of storage. The average encapsulation efficiency of the CO-NLCs was 88.55%. In addition, the CO-NLCs were able to release 20% of eugenol after 2 h, which was lower than the standard (STD)-CO. The CO-NLC at 50 ppm observed the lowest anesthesia (2.2 min), the fastest recovery time (3.3 min), and the most rapid clearance (30 min) in shrimp body biodistribution. The results suggest that the CO-NLC could be a potent alternative nanodelivery platform for increasing the anesthetic activity of clove oil in whiteleg shrimp (P. vannamei).