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A signal detection technique utilising a `lock-in` architecture using the second-order harmonic frequency (2 ωc) applicable to portable optical-biosensor systems is presented. The properties of frequency synthesis and filtering techniques are used to detect a weak signal concealed in intense background noise, such as thermal noise, 1/f noise and shot noise. Using the 2 ωc lock-in, the main signal of interest keeps away from the influence of the 1/f noise, providing a much higher dynamic reserve. In this work, a portable biosensor system using the 2 ωc frequency detection technique is demonstrated. The technique further enhances the minimum detectable range of 20 dB compared to the conventional DC lock-in method under the same conditions.