One of the standard tests for diagnosing AIDS patients, CD4+ counting method is used to measure the number of CD4+ T-lymphocytes per 1 μL of blood and to evaluate the timing of initiation of ART therapy and the effectiveness of treatment in patients with HIV. Although there are several microfluidic point-of-care (POC) devices for monitoring HIV/AIDS, it still has the disadvantage of being expensive to use in developing countries. We invited the 3D helical minichannel-based sample cartridge with a thread-like microgroove formed cylindrical surface and set the particle positioning system with DC motor system based on smartphone. The present study was focused on the particle alignment to monitoring the CD4+ T-cell by spinning the helical minichannel. The helical minichannel system can detect the cells with low sensitivity even in deeper channel than existing 2D flat chamber heights by high-speed spinning of the helical minichannel without automatic focusing module. For proof of concept, the forces acting on the particles in different depth channel were obtained, the particle velocity and particle migration time were obtained and the theoretical and experimental values were compared. As a result, the alignment of the fluorescent bead and CD4+ cells was confirmed by spinning the helical minichannel at 100 μm and 500 μm depth channel and optimized conditions of particle alignment were derived. By using the deep channel, we increased the precision by increasing the amount of sample for cell counting and overcoming the limit of detection by improving the accuracy by aligning the particles. In addition, fluorescence detection performance was confirmed by quantitatively evaluating antigen-antibody reaction conditions for fluorescent labeling of CD4+ cells with low intensity.