A simple and cost-effective, all-electrochemical method to fabricate and assemble single conducting polymer nanowire based biosensors was developed. Polypyrrole (Ppy) nanowires were synthesized by electrochemical polymerization using an alumina template. The single-nanowire chemoresistive sensor device was assembled using ac dielectrophoretic alignment followed by maskless anchoring on a pair of gold electrodes separated by 3 microm. To establish an efficient covalent surface biofunctionalization route, glutaraldehyde (GA) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) chemistries were compared. EDC was established to be the most effective chemistry and was used to surface-functionalize a single Ppy nanowire with cancer antigen (CA 125) antibody to fabricate a nanoimmunosensor for CA 125 biomarker detection and quantification. The immunosensor had excellent sensitivity with a lower detection limit of 1 U/mL CA 125 and dynamic range up to 1000 U/mL in 10 mM phosphate buffer. Furthermore, there was no loss of performance upon exposure to CA 125 in spiked human blood plasma. This demonstrates the clinical importance of these sensors for cancer marker detection with cost benefits and great portability for diagnosis of patients at the point of care.