ObjectiveTo study the influence of different evaporation hardening methods, gelatin concentrations in inter aqueous phase and pH values of PBS on in vitro release modulus of alarelin-loaded microspheres (AR-MS). MethodsThe AR-MS was prepared by double emulsion solvent evaporation method (W/O/W); concentrations of internal aqueous phase with gelatin were 0%, 8% and 16% (W/V); and the volatilized hardening was done with room temperature magnetic stirring and low-temperature vacuum evaporation. Residual method assay was used to determine the accumulated release and burst release of AR-MS in PBS (pH = 4.5, pH = 7.0, pH = 10.0) on day 1, 7, 14, 21, 28, and 35. Scanning electron microscopy (SEM) was used to observe the degradation degrees of AR-MS at each time point. ResultsThe method of volatized organic solvents could affect the release pattern of AR-MS, and the release pattern was also influenced by different concentrations of internal aqueous phase of gelatin. The fastest release rate of AR-MS was seen in medium with pH value being 10.0, complete release was not found in medium with pH being 4.5, and the most appropriate medium for AR-MS release was when pH value being 7.0. ConclusionDifferent evaporation hardening methods can influence the burst release of AR-MS, but has no influence on the overall in vitro release pattern. Different concentrations of internal aqueous gelatin phase can influence the in vitro release pattern, and the medium pH value can also influence the release of AR-MS.