The ongoing outbreak of the novel human coronavirus SARS-CoV-2 (also known as 2019-nCoV) has become a global health concern. Rapid and ease-to-use diagnostic techniques are urgently needed to diagnose SARS-CoV-2 infection.We devised a reverse transcription multiple cross displacement amplification (RT-MCDA) coupled with nanoparticles-based biosensor (BS) assay (RT-MCDA-BS) for rapid, sensitive and specific diagnosis of COVID-19. Two primer sets were designed to target the open reading frame 1a/b (F1ab) and nucleoprotein gene (N) of SARS-CoV-2. A total of 183 clinical samples, including 65 patients with COVID-19 infections and 118 patients with other pathogen infections, were used to testify the assay's feasibility. The diagnosis results were visually reported using BS.The designed assay was performed using a simple instrument which could maintain the reaction in a constant temperature at 64°C for only 35 min. The total procedure of COVID-19 RT-MCDA-BS test could be finished within 1 h. The COVID-19 RT-MCDA-BS could detect down to 5 copies of target sequences. Among 65 clinical samples from the COVID-19 patients, 22 (33.8%) positive results were obtained from face, nasal, pharyngeal and anal swabs via COVID-19 RT-MCDA-BS assay, while rRT-PCR assay only detected 20 (30.7%) positive results in these samples. No positive results were obtained from clinical samples with non-COVID-19 infections.COVID-19 RT-MCDA-BS was a rapid, reliable, low cost and easy-to-use assay, which could provide an attractive laboratory tool to diagnose COVID-19 in multiple clinical specimens, especially for field, clinic laboratories and primary care facilities in resource-poor settings.