Silicon nitride thin films were deposited on the one-sided P-type polished boron-doped silicon wafer substrate via RF magnetron sputtering using stochimetric silicon nitride target at various target-to-substrate distances. Target to substrate spacing, a nonconventional parameter, was varied to optimize the surface roughness and grain size. This optimization provided a normal distribution of homogenous, densely packed silicon nitride thin film free of surface cracks.. Atomic Force Microscopy was employed to explore the accurate surface roughness parameters of Silicon nitride thin films. The surface roughness and grain analysis for all samples exhibited a direct relation to each other and have an inverse correlation with the target to substrate spacing. The surface morphology of Si3N4 was analyzed by the following parameters; average roughness, root-mean square roughness, maximum peak to valley height, ten-point average roughness, skewness, and kurtosis of the line. The surface roughness of silicon nitride films has notable significance in the manufacturing of bio-sensor based on silicon nitride waveguides.