People living with human immunodeficiency virus type 1 (HIV-1), even if successfully treated with a combined antiretroviral therapy, display a persistent inflammation and chronic immune activation, and an increasing risk of developing cardiovascular and thrombotic events, cancers, and neurologic disorders. Accumulating evidence reveals that biologically active HIV-1 proteins may play a role in the development of these HIV-1-associated conditions. The HIV-1 matrix protein p17 (p17) is released and accumulates in different organs and tissue where it may exert multiple biological activities on different target cells. To assess a role of p17 in different HIV-1-related pathological processes, it is central to definitively ascertain and quantitate its expression in a large number of sera obtained from HIV-1-infected (HIV-1+) patients. To this aim, we developed a specific and highly sensitive p17 capture immunoenzymatic assay. Data obtained highlight a heterogeneous expression of p17 in blood of tested patients, with patients who were negative or displayed from low to relatively high p17 blood concentrations (range from 0.05 to 7.29 nM). Moreover, we found that blood p17 concentration was totally independent from the viremic status of the patient. This finding calls for monitoring HIV-1+ patients in order to evaluate a possible correlation between p17 amount in blood and the likelihood of developing HIV-1-related pathological conditions.