In this paper, an indirect method of measuring an analyte concentration in a test solution using the resonant frequency change of a Helmholtz resonator is proposed, using a novel architecture of Helmholtz resonator filled with two kinds of fluids (fixed fluid and test solution). Since the analyte concentration yields changes of density and sound speed of the test solution, the resonant frequency of the proposed Helmholtz resonator is affected by the analyte concentration of the test solution. From this effect, the analyte concentration of the test solution can be measured by the spectrum of acoustic resonance of the Helmholtz resonator. The experiment was done using a 3D-printed Helmholtz resonator system with an acoustic power source and detectors, which is consistent with analytical results and showed that the analyte concentration can be measured with higher sensitivity compared to conventional cantilever-type sensors. As an example application, the possibility of measuring glucose concentration of human blood was demonstrated, showing higher sensitivity and relatively low frequency range compared to previous resonance based methods.