Currently, proniosomes are generating a lot of interest in the field of drug delivery. They are characterized by high stability, extended drug release, ease of manufacturing, and rapid reconstitution and transformation into niosomes. The current study investigated Tramadol HCl (TH) encapsulation into niosomal vesicles using proniosome technology. Tween 80 (T80), Span 80 (S80), Tween 40 (T40), and Span 40 (S40) formed the backbones for niosome preparations. Encapsulation efficiencies (EE), vesicular sizes, in vitro release, and pharmacological activities of niosomal TH preparations were evaluated based on results from the hot plate test. Size distribution analysis showed homogenous vesicles with varied particle size that could be attributed to surfactant type and cholesterol (Chol) content. TH EE increased as a function of the increase in surfactant hydrophilic–lipophilic balance. TH release increased from all formulations as Chol% was increased from 0% to 50%. TH release from niosomal formulations with different surfactant types at 10% Chol increased in a specific order: S80 > S40 > T80 > T40. Following oral administration of TH niosomes to mice, the analgesic effects showed significantly extended effects compared to the TH solution. Oral administration of TH-encapsulated niosomes could be useful for extending the drug's analgesic effects.