A highly sensitive electrocatalytic sensor was designed and fabricated by the incorporation of NiO dope Pt nanostructure hybrid (NiO-Pt-H) as conductive mediator, bis (1,10 phenanthroline) (1,10-phenanthroline-5,6-dione) nickel(II) hexafluorophosphate (B,1,10,P,1,10, PDNiPF6), and electrocatalyst into carbon paste electrode (CPE) matrix for the determination of cysteamine. The NiO-Pt-H was synthesized by one-pot synthesis strategy and characterized by XRD, elemental mapping analysis (MAP), and FESEM methods. The characterization data, which confirmed good purity and spherical shape with a diameter of ⁓ 30.64 nm for the synthesized NiO-Pt-H. NiO-Pt-H/B,1,10, P,1,10, PDNiPF6/CPE, showed an excellent catalytic activity and was used as a powerful tool for the determination of cysteamine in the presence of serotonin. The NiO-Pt-H/B,1,10, P,1,10, PDNiPF6/CPE was able to solve the overlap problem of the two drug signals and was used for the determination of cysteamine and serotonin in concentration ranges of 0.003-200 µM and 0.5-260 µM with detection limits of 0.5 nM and 0.1 µM, using square wave voltammetric method, respectively. The NiO-Pt-H/B,1,10,P,1,10,PDNiPF6/CPE showed a high-performance ability for the determination of cysteamine and serotonin in the drug and pharmaceutical serum samples with the recovery data of 98.1-103.06%.