Mitogen-activated protein kinase (MAPK) cascade reaction is one of important and complex signal networks involved in plant growth and development, hormone and stress response. As one of its main members, mitogen-activated protein kinase kinase (MAPKK) is located in the middle of the cascade reaction and plays a key role in signal collection and divergence. There were shown that potato (Solanum tuberosum L.) StMAPKK1 (PGSC0003DMT400000744) gene responded to drought stress. Therefore, StMAPKK1 gene was firstly selected to screen its interacting protein. In this study, the bait vector pGBKT7-StMAPKK1 was constructed by homologous recombination and used to screen potato cDNA library by yeast (Saccharomyces cerevisiae) two-hybrid system. Five StMAPKK1-interacting proteins, hydrolase (hydrolyzing O-glycosyl compound), RING-H2 subgroup RHE protein, cyanate hydratase, ARF GTPase activator, and a C2 domain-containing protein, were obtained through this screening and were identificated by bioinformatics analysis, and the interaction was verified by small-scale hybridization verification. The results provided theoretical basis for further study on the signal pathway and biological function of potato StMAPKK1.