In this study, nanovesicles were developed for brimonidine tartrate by film hydration technique and dispersed in viscous carbopol solution for ocular delivery. Scanning electron microscopy revealed spherical shape of the vesicles. As high as 32.27% drug entrapment efficiency was achieved depending upon the surfactant/cholesterol molar ratio (7:4 to 7:8). The vesicles were in the size range of 298.0–587.9 nm. Release study showed a biphasic drug-release pattern for the lyophilized vesicular formulation in buffered saline solution, i.e., initial burst release followed by gradual release over the period of 8 h. On contrary, the isolated vesicles reduced the burst effect in 3 h by two to three times and the drug release was comparatively slower at the intermediate ratio in both cases. With variation in cholesterol content, the drug release followed either first order or Higuchi’s kinetics. Physically the lyophilized vesicular formulations were more stable at refrigerated temperature. DSC and X-RD analyses indicated loss of drug crystallinity in the vesicles. FTIR spectroscopy did not reveal any interaction between drug and excipients. The lyophilized formulation showed better ocular hypotensive activity than marketed drops on albino rabbits and in vivo efficacy was sustained up to 7.5 h. Furthermore, the formulation was found to be non-irritant to the rabbit eye. Hence, the lyophilized vesicles, when dispersed in viscous carbopol solution, had the potential in reducing dosing frequency and could improve patient compliance.