Abstract: To prepare thymosin alpha-1 (Tα₁) loaded injectable sustained release microspheres and to evaluate its release behavior, bioactivities in vitro as well as its pharmacodynamics in vivo, Tα₁ loaded microspheres was prepared with poly(lactic-co-glycolic acid) (PLGA) as carrier material by double emulsion (W/O/W) method. Physical and chemical properties of microspheres, such as mean diameter, morphology and drug loading were evaluated. The release behavior and its influencing factors were evaluated by HPLC determination. The bioactivity of Tα₁ in the course of encapsulation process and in vitro release ware evaluated by CCK-8 method. The ratio of CD₄⁺/CD₈⁺ in blood was determined with flow cytometry and the pharmacodynamics of Tα₁ loaded microspheres was evaluated by the change of CD₄⁺/CD₈⁺. Microspheres with good shape and dispersive quality were prepared. The drug entrapment efficiency of two optimizing prescriptions containing 5% NaCl and 10% glucose as outer water phase were 87.8% and 90.2%, respectively. The cumulated release in one month is up to 90%. The bioactivity of Tα₁ was conserved with glucose as outer water phase, but in the course of in vitro release, the specific activity of Tα₁ in the microspheres decreased a little. Tα₁ microspheres can increase significantly the immunity of immuno-suppressed rats. Tα₁ can be encapsulated in injectable microspheres to yield one-month continuous release when using biodegradable polymers PLGA as carrier material, and this technique will have a favorable perspective in the near future.