Helmholtz resonators (HR) have been proven to have feasibility for sensor applications with good performance. However, for biosensor application, when the amount of test sample is limited, the operating frequency tends to be very high, which may bring challenges of resonance excitation and frequency measurement. In this paper, a modified HR was proposed for biosensor application at a lower frequency, by designing the neck of the HR as a type of an Archimedes spiral, which can increase the neck length as much as possible without occupying much space. The resonant frequency of the proposed HR was derived by treating it as an HR with a straight neck with the equivalent length. An analytical example was calculated and verified by acoustic finite element analysis, and the results clearly showed that the frequency decreases in comparison to that of the conventional HR. Based on the underlying theory, the measurement principle was proposed and the experiment setup was established using a 3D-printed structure. The experiment was done using test solutions with different glucose concentrations, whose results showed consistency with the analytical results and noticeable frequency increases with the glucose concentration. Prospective results of the proposed HR after miniaturization show an operating frequency around 3 kHz when the amount of test sample is 0.1 mL, which will facilitate use of the common acoustic power source and acoustic pressure sensors in the sensing system.