This work represents experimental and simulation analysis of photonic crystal waveguide (PCW)-based biosensor structures, which is used for detection of the Escherichia coli (E. coli) cell. A method is adopted for E. coli culture to measure length, diameter, and refractive index to finalize the structural design and to verify the suitability of PCW as a biosensor. This method is tested using DH5α strains of E. coli. The typical precisions of measurements are varied in ranges from 1.132 to 1.825 μm and from 0.447 to 0.66 μm for pathogen’s length and diameter, respectively. The measured distribution of samples over length and diameter are in correlation with the measurements performed by scanning electron microscope. After obtaining average length and diameter of cylindrical shaped E. coli cell, we consider these values for simulation analysis of designed PCW biosensor. E. coli cell is trapped in the middle of the PCW biosensor having three different types of waveguides, i.e., gallium arsenide/silicon dioxide (GaAs/SiO2), silicon/silicon dioxide (Si/SiO2), or silicon nitride/silicon dioxide (Si3N4/SiO2) to observe the maximum resonance shift and sensitivity. It is observed from the simulation data analysis that GaAs/SiO2 is the preferred PCW biosensor for the identification of E. coli.