To enhance the transdermal delivery performance of recombinant human collagen Ⅲ (RHC), glycerosomes were constructed from soybean lecithin (for short lecithin), glycerol and Tween 80, and subsequently used for encapsulation of RHC to form RHC-loaded glycerosomes (RHC glycerosomes). A Folch-based high-performance liquid chromatography method (Folch-HPLC method) combining the Folch extraction protocol with size exclusion chromatography was established to measure the RHC encapsulation efficiency. The effects of glycerol and Tween 80 mass concentrations on the particle size and polydispersity index (PDI) of RHC glycerosomes were investigated by multi-angle particle size and high sensitivity Zeta potential analyzer, while the influence of glycerol and Tween 80 mass concentration on the micro-viscosity and stabilization mechanism of RHC glycerosomes was explored via fluorescence probe analysis. The transdermal delivery capability of RHC glycerosomes was evaluated by confocal laser scanning microscopy (CLSM), with its irritancy assessed by chorioallantoic membrane assay in chicken embryos. The results demonstrated that the Folch-HPLC method effectively removed interference from lecithin while retained the RHC, achieving an RHC recovery rate of 100.44%±0.84%. Under the conditions of lecithin mass concentration 150 g/L, RHC mass concentration 2 g/L, glycerol mass concentration 250 g/L, and Tween 80 mass concentration 25 g/L, the prepared RHC glycerosomes appeared as a yellow, clear, and transparent solution, showed the best stability, displayed a particle size of (92.34±1.22) nm, a PDI of 0.233±0.01 and an encapsulation efficiency of 12.78%±0.13%. Compared with RHC solution (phosphate buffer solution as solvent), RHC glycerosomes exhibited significantly enhanced skin penetration, effectively promoted the transdermal delivery of RHC, and showed no irritation in chorioallantoic membrane.