Biomolecules play important roles in physiological functions and pharmacological characteristics of human body. Uric acid (UA) is the end product of purine. Dopamine (DA) is a neurotransmitter of catecholamine group. l-tryptophan is an essential amino acid that can be metabolized to neuroactive substances. Pyridoxine is a water-soluble vitamin playing an important role in nervous system. The abnormalities in their concentration levels led to a wide range of significant mental and physical illnesses. Thus, electrochemical analysis of these analytes on an array system would be beneficial from clinical or scientific points of view. This work was aimed at the development of practical sensor array for determination of multiple analytes on a single sensing platform using individually addressable microelectrodes. The occurrence of adsorption–desorption phenomenon on the surface of palladium microelectrode array (Pd MEA) printed on the silicon wafer through photolithography was exploited for electro-oxidation of UA, DA, l-tryptophan and pyridoxine. The sensing of electroactive UA was done using carbon nanotubes(CNTs) grown Pd MEA as a working electrode, while selectivity for other analytes was achieved by the modification of CNTs/Pd MEA through electrodeposition of poly(l-lysine) (poly(l-lysine)/CNTs/Pd MEA) for DA sensing, poly(l-arginine) (poly(l-arginine)/CNTs/Pd MEA) for l-tryptophan sensing and reduced graphene oxide (rGO/CNTs/Pd MEA) for pyridoxine sensing. The electrochemical differential pulse voltammetry (DPV) analyses reveal excellent linearity in the concentration ranges of 50–6000 µmol/L, 2–8000 µmol/L, 20–15,000 µmol/L, and 10–5000 µmol/L with detection limits of 15.0, 0.5, 10.0, and 1.0 µmol/L for UA, DA, l-tryptophan, and pyridoxine, respectively. The proposed multiple analytes sensor has shown very high sensitivities of 140, 9580, 2280, and 940 µA·(µmol·L⁻¹)⁻¹·cm⁻² for UA, DA, l-tryptophan, and pyridoxine sensing, respectively. Further, accuracy and reliability of the fabricated sensor were also tested in real samples.