Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient protein research and has been used for macromolecular assembly, unnatural amino acid embedding, glycoprotein production, and more. To realize the construction of an efficient eukaryotic CFPS platform with the advantages of low cost and short time, a CFPS system based on the yeast Pichia pastoris was built in this study. The internal ribosomal entry site (IRES) can independently initiate translation and thus promote protein synthesis. The Kozak sequences can facilitate translation initiation. Therefore, the screening of IRES and its combination with Kozak was performed, in which cricket paralysis virus (CRPV) exhibited as the best translation initiation element from 14 different IRESs. Furthermore, the system components and reaction environment were explored. The protein yield was nearly doubled by the addition of RNase inhibitor. The cell extract amount, energy regeneration system (phosphocreatine and phosphocreatine kinase), and metal ions (K and Mg) were optimized to achieve the best protein synthesis yield. This P. pastoris CFPS system can extend the eukaryotic CFPS platform, providing an enabling technology for fast prototyping design and functional protein synthesis.