A fresh water fish bio-impedance measurement system was constructed based on LabVIEW and bio-impedance theory, and the sensor was developed by the four electrodes structure. The relation between the excitation frequency and fish bio-impedance was researched by sweeping frequency driving method. The experiment also investigated the relation between measuring direction and bio-impedance, the relation between electrode structure and bio-impedance, and the relation between measuring position and bio-impedance, which supports the theory foundation for constructing the measurement system of freshwater fish freshness. The results indicated that the fish bio-impedance amplitude decreases and phase angle increases as the frequency increases, and the bio-impedance measured along the lateral line was bigger than the bio-impedance measured vertical to the lateral line, which conforms to biological tissue's anisotropism. Different measured position results in different bio-impedance, and at the same measuring position, different decay time gets different bio-impedance. The composite electrode measuring gill of fish is suitable for evaluation of fresh water fish freshness due to its stable and accurate test signal. 