Mycoplasma hyorhinis is a normal flora in swine respiratory tract and also often found in multiple human tumor tissues, which is considered to be highly correlated with human tumors. Due to the detection of Mycoplasma hyorhinis mainly relies on PCR-based assay at present, thus it is critical for developing a novel assay for rapid detection and providing support diagnosis evidence. In our work, we screened and characterized a high affinity aptamer zyb1 that can recognize Mycoplasma hyorhinis based on infectious cell-SELEX. On this basis, we developed a lateral flow strip assay by using zyb1 and another aptamer AP15-1 to form a sandwich-type aptasensor. Using this new lateral flow strip assay biosensor, Mycoplasma hyorhinis could be detected within the detectable limit as low as 1 × 10³ CCU/mL. Therefore, our study successfully developed a convenient and effective lateral flow strip for Mycoplasma hyorhinis detection and demonstrated the potential of utilizing aptamer for the development of point-of-care testing products for mycoplasma detection.