In this work, we report on the design of a wide-band digital lock-in amplifier (DLIA) of up to 65 MHz and its application for electrical impedance measurements in microfluidic devices. The DLIA is comprised of several dedicated technologies. First, it features a fully differential analog circuit, which includes a preamplifier with a low input noise of 4.4 nV/√Hz, a programmable-gain amplifier with a gain of 52 dB, and an anti-aliasing, fully differential low-pass filter with −76 dB stop-band attenuation. Second, the DLIA has an all-digital phase lock loop, which features a phase deviation of less than 0.02° throughout the frequency range. The phase lock loop utilizes an equally accurate period-frequency measurement, with a sub-ppm precision of frequency detection. Third, a modified clock link is implemented in the DLIA to improve the signal-to-noise ratio of the analog-to-digital converter affected by clock jitter of up to 20 dBc. A series of measurements were performed to characterize the DLIA, and the results showed an accurate performance. Additionally, impedance measurements of standard-size microparticles were performed by frequency sweep from 300 kHz to 30 MHz, using the DLIA in a microfluidic device. Different diameters of microparticle could be accurately distinguished according to the relative impedance at 2.5 MHz. The results confirm the promising applications of the DLIA in microfluidic electrical impedance measurements.