A leakage-free disposable package for biosensors was developed to improve the accuracy of electrochemical measurement. The traditional biosensor packaging such as diabetes test strip is not appropriate for the biosensor immersed in the chemical solution since it is easier to permeate through the layer between the substrate and the sensor chip to increase the errors of electrochemical impedance spectroscopy (EIS) measurement. It is possible to provide the incorrect diagnostic results to the patients. In this study, a novel package was designed and fabricated to solve this critical issue. Combined with the package-level flip chip and the assembly-level drilling technologies, the main advantage of the package is that the specific quantity of the chemical solution can be guided precisely into the cavity to react with the controlled region on the top side of the biosensor to stabilize the electrochemical reaction, and thus to increase the accuracy of the impedance measurement. The EIS results present the stable and smooth curves to verify the appropriate package design. Compared to the previous design by authors using traditional way of the front side gluing to the biosensor chip, the backside gluing is better to prevent the solution to flow into the sensor body, substrate, and the electrode so that it can increases the diagnostic accuracy corresponding to the sensitivity and specificity.