Since the beginning of the COVID-19 pandemic, accumulating mutations have led to marked changes in the genetic sequence of SARS-CoV-2. Of these, mutations in the spike (S) protein can alter the properties of the virus, particularly transmissibility and antigenicity. However, it is difficult to detect antigenic variants of the SARS-CoV-2 S protein by immunoassay. Here, we developed an ACE2-based biosensor designed to detect both SARS-CoV-2 S1 mutations and neutralizing antibodies. In "binding mode", the biosensor works by detecting binding of the S protein to an immobilized ACE2 receptor. The ACE2-based biosensor was able to detect S1 proteins of the alpha (500 pg/mL) and beta variants (10 ng/mL), as well as wild-type S1 (10 ng/mL), of SARS-CoV-2. The biosensor distinguished wild-type SARS-CoV-2 S1 from the S1 alpha and beta variants via color differences. In addition, a slight modification to the protocol enabled the ACE2-based biosensor to operate in "blocking mode" to detect neutralizing antibodies in serum samples from COVID-19 patients. Therefore, the ACE2-based biosensor is a versatile test for detecting wild-type S1, S1 mutants, and neutralizing antibodies against SARS-CoV-2. This approach to targeting both the mechanism by which SARS-CoV-2 enters host cells and the subsequent adaptive immune response will facilitate the development of various biosensors against SARS-CoV-2.