Background: Cancer stem cells (CSCs) are of great diagnostic importance due to their involvement in tumorigenesis, therapeutic resistance, metastasis and relapse. Method: In this work, a sensitive electrochemical cytosensor was successfully established to detect HT-29 colorectal cancer stem cells based on a nanocomposite composed of mesoporous silica nanoparticles (MSNs) and platinum nanoparticles (PtNPs) using a simple and fast electrodeposition technique on a glassy carbon electrode (GCE). Results: According to SEM images, the PtNPs nanoparticles formed on the MSNs substrate are about 100 nm. As expected, high-rate porosity, increased surface-to-volume ratio, provides appropriate local electron transfer rate and suitable platform for the efficient formation of PtNPs. These features allow direct and stable binding of biotinylated monoclonal antibody of CD133 to streptavidin (Strep) and the subsequent availability of active sites for CSCs identification. Differential pulse voltammetry (DPV) results show that close interaction of CD133+ cells with monoclonal antibodies reduces charge transfer and electrical current, as confirmed by square wave voltammogram (SWV). Based on the recorded current versus number of CSCs, we noted that our developed system can sense CSCs from 5 to 20 cells/5 μL. Conclusions: As a proof of concept, the designed nanobiocomposite was able to specifically detect CD133+ cells compared to whole HT-29 cells before magnetic activated cell sorting (MACS) process.