Objectives To develop a novel thrombopoietin (TPO) analog by fusing the tandem TPO mimetic peptide (TMP-TMP) to human serum albumin (HSA) and performing functional expression of recombinant fusion protein HSA-TMP-TMP. Results After optimizing the fusion orientation in shake-flask culture, HSA-TMP-TMP was expressed at 0.4 g/l in Pichia pastoris grown in a 20 l bioreactor, during which pH was controlled at 5 by addition of NH4OH and citric acid. The fusion protein significantly activated signal transducer and activator of transcription-mediated transcription in TPO receptor-dependent manner, which was demonstrated by a luciferase reporter assay. Following subcutaneous administration, HSA-TMP-TMP effectively stimulated the platelet production in healthy mice in a dose-dependent manner. Conclusion Successful expression of HSA-TMP-TMP fusion protein in P. pastoris was achieved and the recombinant HSA-TMP-TMP is a promising TPO analog.