The outbreak of coronavirus disease 2019 (COVID-19) has overwhelmed the global economy and human well-being. On account of the sharp increase in test demand, there is a need for an accurate and alternative diagnosis method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, with the aim to specifically identify the trace SARS-CoV-2 S1 glycoprotein, we developed a high-sensitivity and high-selectivity diagnostic method based on the targeted parallel reaction monitoring (PRM) assay of eight selected peptides. This study emphasizes the outstanding detection sensitivity of 0.01 pg of the SARS-CoV-2 S1 glycoprotein even in the interference of other structural proteins, which to our knowledge is the current minimum limit of detection for the SARS-CoV-2 S1 glycoprotein. This technology could further identify 0.01 pg of the SARS-CoV-2 S1 glycoprotein in a spike pseudovirus, revealing its practical effectiveness. All our preliminary results throw light on the capability of the mass spectrometry-based targeted PRM assay to identify SARS-CoV-2 as a practicable orthogonal diagnostic tool. Furthermore, this technology could be extended to other pathogens (e.g., MERS-CoV S1 protein or SARS-CoV S1 protein) by quickly adjusting the targeted peptides of MS data acquisition. In summary, this strategy is universal and flexible and could be quickly adjusted to detect and discriminate different mutants and pathogens.