In this work, we offered a novel and sensitive electrogenerated chemiluminescence (ECL) biosensing system for detection of p16(INK4a )gene using the functional paste-like nanofibers composites-modified screen-printed carbon electrode (SPCE). The paste-like nanofibers composites (PG/GR/CS) which were comprised of the electrospun nanofibers (PG, the graphene (GR) doped polycaprolactam 6 (PA6) were prepared via one-step electrospinning), graphene (GR) and chitosan (CS) were served as the nanosized backbones for pyrrole (Py) electropolymerization. The functional paste-like nanofibers composites (PG/GR/CS/PPy) used as a substrate for dsDNA (hybridization reaction of ssDNA1, p16(INK4a) gene and the Ru(bpy)(3)(2+)/silver nanoparticles (AgNPs) doped gold (Au) core-shell luminescent composite nanoparticles labeled ssDNA2 (RuAg@AuNPs-ssDNA2)) immobilization. Under optimal conditions, a linear relationship between ECL intensity and p16(INK4a )gene concentration was found in a range of 0.1 pM-1 nM with the detection limit of 0.05 pM (S/N = 3). This ECL biosensor based on the PG/GR/CS/PPy-modified SPCE demonstrated excellent electrochemical performance for the detection of p16(INK4a) gene and this platform can be used for the determination of various analytes.