A spinal cord injury is damage to the nerves and cells that receive and provide a signal from the brain to the rest of the body. Spinal injury causes changes in movement, sensation, and strength, affect the body functions near the injury site, and may lead to paralysis. S100β was found as a suitable biomarker for identifying spinal cord injury and its causing problem. Herein, S100β immunoassay was developed on interdigitated electrode sensor to diagnose spinal cord injury. For effective anti-S100β antibody immobilization, the antibody was premixed with 3-Aminopropyl)triethoxsilane and then attached to the hydroxylated interdigitated electrode surface. This method of antibody immobilization enhanced the antibody attachment two-times than the method without premix. Antibody-attached surfaces increased current responses as S100 concentrations increased, and the limit of detection was seen to be 1 pg/mL on the linearity until 3000 pg/mL at an R² value of 0.9907 [y = 7x - 6.4667]. Further, biofouling experiments with glial fibrillary acidic protein and γ-aminobutyric acid failed to enhance the current response, indicating the specific detection of S100β. This immunoassay identifies S100β at its lower level and helps to diagnose spinal cord injury and its related problem.