The Coronavirus Disease 2019 (COVID-19) has created serious risks to human health and public safety. Hence, there is a pressing demand for uncomplicated and precise diagnostic assays to ensure accurate identification of the infection. SARS-CoV-2, like other coronaviruses, is classified as a single-stranded, positive-sense RNA virus with four key structural proteins: the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The spike protein holds significant importance in early diagnosis of SARS-COV-2 infection due to its role in viral attachment, fusion, and cellular entry. In the current study, the diagnostic performance of SARS-CoV-2 Spike Protein S1 RBD was evaluated. Clinical samples (n =75) (nasopharyngeal and blood specimens) were collected from confirmed infected patients using reverse transcription polymerase chain reaction RT-PCR. In addition, 25 healthy participants were included as controls. Routine laboratory markers were evaluated for all participants and the SARS-CoV-2 Spike Protein S1 RBD was determined using sandwich ELISA for all participants. The result showed that the spike protein level demonstrated a statistically significant difference (p < 0.0001) in COVID-19 patients compared to healthy participants. Furthermore, the variables D-Dimer and CRP demonstrated a statistically significant difference (p < 0.0001) with the existence of COVID-19. In contrast, the analysis revealed no differences regarding LDH, body mass index, or gender among the studied groups. In addition, there was a significant positive correlation between D-dimer (r = 0.57, p<0.0001) and spike antigen. Spike antigen was the most effective biomarker in distinguishing COVID from healthy participants, with an AUC of 0.99, sensitivity of 98%, and specificity of 94%. Additionally, D-dimer has a sensitivity of 93%, a specificity of 92%, and an AUC of 0.96, and CRP has a sensitivity of 87%, a specificity of 80%, AUC=0.88. And finally, ferritin has a sensitivity of 61%, a specificity of 64%, and an AUC of 0.64. SARS-COV-2.Spike antigen can be used as a suitable diagnostic test for identifying COVID-19 infection with higher sensitivity and specificity.