Many studies in the field of cold plasma and its application in medicine and biology have been done recently. Some of them reported that cold atmospheric pressure plasma jet (APPJ) discharge accelerates blood clotting because factor VIII (FVIII) is a plasma glycoprotein that may play a role in the coagulation cascade. In this study, we aim to assess the effect of APPJ discharge on factor VIII antigen (FVIII:Ag) in healthy and hemophilic patients. Therefore, the variations of FVIII:Ag in healthy plasma samples and hemophilic plasma samples at the times of 0, 4, 8 and 16 s treatment with APPJ were measured by ELISA technique. Additionally, activated partial thromboplastin time (APTT) test was used to check the blood coagulation rate at different treatment times. Our findings showed that the amount of FVIII:Ag in healthy samples was high to low in seconds 4 > 8 > 16 > 0. The APTT results showed that APPJ increases the blood clot formation, but in hemophilic plasma samples, APPJ reduces the clot formation speed and FVIII:Ag concentration. Accordingly, the APPJ discharge elicited a slight increase in FVIII:Ag concentration in short-term treatment, which could be involved in blood coagulation. Moreover, the effects of APPJ discharge on other coagulation factors need further studies.