Objective:Establishment of glucokinase activator screening method using heterologous expression of eukaryon system. Methods:The pPIC9K vector was inserted a fragment of human liver glucokinase. The plasmid of pPIC9KGK was identified by restriction enzyme and sequencing. Then the plasmid was transfected to pichia pastoris via methanol induction. They produced the human liver recombinant GK protein,which was further detected by SDS-PAGE. The recombinant GK protein had enzymatic activity and was used in GK activator screening method. Using this method, lots of compounds to find GK activators were screened and a lead-compound LGK-01 was found. Results:Human liver recombinant GK protein was expressed in pichia pastoris, and the protein had enzymatic activity. Conclusion:GK activator screening method was established successfully in vitro.