A novel modified electrode was fabricated by a mixed-valent nickel hexacyanoferrate (NiHCF) and poly 1,5-diaminonaphthalene (PDAN) hybrid at glassy carbon (GC) electrode. The obtained NiHCF/PDAN/GC modified electrode was characterized using cyclic voltammetry (CV) technique and scanning electron microscope (SEM). This electrode showed excellent catalytic properties toward the electrooxidation of ascorbic acid (A.A), dopamine (D.A) and uric acid (U.A) in 0.1 M NaCl solution using CV and square wave voltammetry (SWV) techniques. The NiHCF/PDAN/GC modified electrode exhibits high sensitivity, selectivity and stability making it a suitable sensor for the simultaneous detection of A.A, U.A and D.A in single and ternary mixture solutions. Different analytical parameters such as low detection limit (LOD), low quantification limit, correlation coefficient (R) and linear dynamic range were reported and discussed. The NiHCF/PDAN/GC modified electrode exhibits linear responses to A.A, D.A and U.A in the range 600–1000, 600–1000 and 600–1000 µM, respectively. The LOD for A.A, D.A and U.A were 0.036, 0.034 and 0.037 µM, respectively. The analytical behavior of this sensor had been evaluated for the detection of A.A and U.A in human serum and urine samples with satisfactory results.