Laser-scribed graphene (LSG), a promising electrode material has attracted special research interest in recent years. Here, the fabricating process-electrochemical property correlation of laser-scribed graphene (LSG) devices was discussed emphatically and a pertinent optimization was performed to achieve better electroanalytical performance. Experiment results indicated that the laser scribing technique possessed great process latitude and reducing laser power and scribing speed facilitated fabricating high-quality graphene electrodes. Benefiting from its binder-free 3D porous network structure and high active/geometric area ratio, the optimized LSG electrode was superior to the screen-printed carbon electrode (SPCE) on electrochemical performance in the [Fe(CN)] redox system. Integrating the LSG electrode with a homemade hand-held detector, a portable electrochemical sensing platform with smartphone readout was developed. It realized a specific detection of HO (linear range: 0.02-3.4 mM, sensitivity: 24.56 μA mM cm), glucose (linear range: 0.04-4.0 mM, sensitivity: 16.35 μA mM cm) by directly decorating biological enzymes without artificial redox mediator and featured a satisfactory comprehensive performance. The constructed immunosensor for tumor necrosis factor-α exhibited a wide linear range (2-500 pg mL) and a 4.3-fold enhancement in sensitivity compared with that of SPCE. With satisfactory selectivity, reproducibility, and sensitivity, the developed smartphone-based electrochemical sensing platform held great promise in accurate detection on the spot. This work also provided a significant reference for tailoring binder-free carbonaceous electrode materials toward the desired application.