Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia is a newly recognized disease, and its diagnosis is primarily confirmed by routine RT-PCR detection of SARS-CoV-2. However, we report a confirmed case of SARS-CoV-2 pneumonia with routine RT-PCR negative. This case has been finally diagnosed by Nanopore sequencing combined with antibody of SARS-CoV-2. Simultaneously, the ORF and NP gene variation of SARS-CoV-2 were found. This case has highlighted that false negative results could be present in the routine RT-PCR diagnosis, especially with virus variation. At the moment, Nanopore pathogen sequencing and antibody detection have been found effective in clinical diagnosis.