Neutral DNA analogs as probes for the detection of target oligomers on the biosensors based on the field-effect transistor (FET) configuration feature advantages in the enhancement of sensitivity and signal-to-noise ratio. Herein, we used phosphate-methylated nucleotides to synthesize two partially neutralized chimeric DNA products and a fully neutralized DNA sequence and adopted a regular DNA oligomer as probes on the polycrystalline silicon nanowire (NW) FET devices. The sequences of two neutralized chimeric DNAs close to the 5′ end were alternately modified with the phosphate-methylated nucleotides, and all probes were immobilized via their 5′ end on the NW surface. The non-specific-to-specific binding ratio indicated that the two 5′-end partially neutralized chimeric DNAs featured better performance than the regular and fully neutralized DNA oligomers. The partially neutralized probe design reduces the ionic strength needed for hybridization and increases the Debye length of detection, thus promoting the detection sensitivity of FET and achieving the limit of detection of 0.1 fM. By using an appropriate probe design, applying DNA oligomers with embedded phosphate-methylated nucleotides in the FET biosensors is a promising way for gene detection with high sensitivity and specificity.