AbstractThe current outbreak of monkeypox virus (MPXV) has become a public health emergency of international concern that highlights the need for rapid, sensitive MPXV diagnostic assays. Here, we combined isothermal multiple cross displacement amplification (MCDA) with nanoparticle‐based lateral flow biosensor (LFB) to devise a diagnostic test for the diagnosis of MPXV infection (called MPXV‐MCDA‐LFB) and differentiation of West and Central African MPXV isolates. The MPXV‐MCDA‐LFB protocol conducts isothermal MCDA reaction for DNA templates followed by LFB detection of preamplification target sequences. Two MCDA primer sets were designed targeting the D41L and ATI genes of Central and West African MPXV isolates, respectively, and the optimal condition of two MCDA reactions was 64°C for 30 min. The two MCDA reactions were decoded by LFB, which was devised for detecting three targets, including two amplicons yielded from two MCDA reactions and a chromatography control. Thus, the MPXV‐MCDA‐LFB assay could be completed within 50 min including rapid template preparation (15 min), MCDA reaction (30 min) and reporting of result (<5 min). The MPXV‐MCDA‐LFB method is very sensitive and can detect the target genes (D14L and ATI) with as low as five copies of plasmid template per reaction and 12.5 copies of pseudotyped virus in human blood samples. The MPXV‐MCDA‐LFB assay does not cross‐react with non‐MPXV templates, validating its specificity. Therefore, the MPXV‐MCDA‐LFB assay developed here is a useful tool for rapid and reliable diagnosis of MPXV infection.