The analgesic effects of neurotoxin (NT) found in snake venom are well-documented. However, their clinical use is hindered by the pain experienced after intramuscular injection. Dissolving microneedles (DMNs) offer a way for transdermal drug delivery by directly penetrating the stratum corneum (SC) with micron-sized needles. Importantly, the penetration depth of DMNs does not stimulate nerve endings associated with pain, rendering this method painless during drug administration. In this study, we prepared NT-loaded DMNs (DMN-NT) using different materials and evaluated their properties and analgesic effects. The manufactured DMN-NT exhibited a square pyramid morphology, with a length of 475 μm, a width of 300 μm, and a uniformly smooth surface. The DMN-NT demonstrated favorable mechanical properties, capable of penetrating hairless mice skin to a depth of 70-80 μm. Additionally, they showed a sustained-release profile upon transdermal penetration through the SC, enabling a continuous release of the drug for a duration of 4 h. The analgesic effects of the DMN-NT were found to be adequate in both peripheral and central analgesia tests. In conclusion, the use of DMN-NT enables painless transdermal drug delivery and prolongs the duration of the analgesic effect of NT.