A novel, cheap, disposable and single-use nanoparticles-based nanochannel platform assembled on a flexible substrate for label-free immunosensing is presented. This sensing platform is formed by the dip-coating of a homogeneous and assembled monolayer of carboxylated polystyrene nanospheres (PS, 200 and 500 nm-sized) onto the working area of flexible screen-printed indium tin oxide/polyethylene terephthalate (ITO/PET) electrodes. The spaces between the self-assembled nanospheres generate well-ordered nanochannels, with inter-PS particles distances of around 65 and 24 nm respectively. The formed nanochannels are used for the effective immobilization of antibodies and subsequent protein detection based on the monitoring of [Fe(CN)6]4– flow through diffusion and the decrease in the differential pulse voltammetric signal upon immunocomplex formation. The obtained sensing system is nanochannel-size dependent and allows human immunoglobulin G (IgG) (chosen as a model analyte) to be detected at levels of 580 ng/mL. The system also exhibits an excellent specificity against other proteins present in real samples and shows good performance with a human urine sample. The developed device represents an integrated and simple biodetection system which overcomes many of the limitations of previously reported nanochannels-based approaches and can be extended in the future to several other immuno and DNA detection systems.