The counting of CD4+ T lymphocytes is a clinical parameter used for AIDS diagnosis and follow-up. As this disease is particularly prevalent in developing countries, simple and affordable CD4 cell counting methods are urgently needed in resource-limited settings. This paper describes an electrochemical magneto-actuated biosensor for CD4 count in whole blood. The CD4+ T lymphocytes were isolated, preconcentrated and labeled from 100μL of whole blood by immunomagnetic separation with magnetic particles modified with antiCD3 antibodies. The captured cells were labeled with a biotinylated antiCD4 antibody, followed by the reaction with the electrochemical reporter streptavidin-peroxidase conjugate. The limit of detection for the CD4 counting magneto-actuated biosensor in whole blood was as low as 44cellsμL−1 while the logistic range was found to be from 89 to 912cellsμL−1, which spans the whole medical interest range for CD4 counts in AIDS patients. The electrochemical detection together with the immunomagnetic separation confers high sensitivity, resulting in a rapid, inexpensive, robust, user-friendly method for CD4 counting. This approach is a promising alternative for the costly standard flow cytometry and suitable as diagnostic tool at decentralized practitioner sites in low resource settings, especially in less developed countries. •There is an urgent need for novel affordable alternatives to flow cytometry for CD4 count to monitor the AIDS disease and the treatment in low resource settings.•An electrochemical magneto-actuated biosensor for CD4+ T lymphocytes count in whole blood is presented, with a LOD of 44cellsμL−1 covering the clinical range of CD4+ lymphocytes in AIDS diagnosis and follow up.•The electrochemical magneto biosensors offer an exciting alternative in resource-scarce settings, as rapid, cost-effective devices that can be handled for unskilled personnel at the community care level.