A novel electrochemical cytosensor was developed based on PAMAM and a folic acid (FA) modified glassy carbon paste electrode (GCPE) where HeLa cells were utilized as model cancer cells. For this purpose, gold nanoparticles (AuNp), cysteamin (Cys), glutaraldehyde (Glu), PAMAM and FA were immobilized onto the GCPE respectively. After the characterization of the GCPE/AuNp/Cys/Glu/PAMAM/FA cytosensor and optimization of the experimental parameters, analytical characteristics were examined. The linear range was found between 10² cells per mL and 10⁶ cells per mL. The LOD value was calculated as 100 cells per mL with RSD value of 1.55% (for 5.0 × 10⁴ HeLa cells per mL (n = 3)). The selectivity of the GCPE/AuNp/Cys/Glu/PAMAM/FA cytosensor was tested by using the folate negative cell line A549. The cytosensor's performance was also compared with similar previous studies. As a result, a selective, sensitive and practical system was developed.