Due to the high specific surface area of microbubble-based systems, the concept of gas-liquid separation has successful applications in many fields, such as oil-water separation, algal harvesting, micro-extraction, membrane pretreatment, and water treatment. Gas holdup is an important parameter in such systems. However, the conventional measurement methods for the macrobubble system may not be directly applicable to the microbubble system due to small bubble size and low gas holdup. In this study, an ultrasound-conductivity method under non-isokinetic sampling conditions was developed to measure gas holdup in the microbubble-based gas-liquid system. The measurement setup consists of a sampling probe, a bubble coalescence unit, and a conductivity measurement unit. A key feature of the setup is a bubble coalescence unit to convert microbubbles to macrobubbles for conductivity measurement. The results showed that the bubble coalescence unit, made of an ultrasonic bath and a bubble-coalescence cell, was successful in forming macrobubbles so that accurate conductivity measurements could be made. Under non-isokinetic sampling conditions, the relationship between the extraction parameter (the gas volume fraction in the sampling probe) and the true gas holdup was established in flotation columns. The results indicate that the relationship does not depend on other factors, such as sampling probe orientation and flotation column diameter. Therefore, the developed ultrasound-conductivity method has great potential in microbubble-based gas-liquid system.