The demand for on-chip cancer cell separation in biomedical, bio-sensor, bio-detection, personalized medicine, and clinical diagnostic fields has resulted in a variety of studies focusing on this theme. Accurate, high-throughput, and non-invasive separation of individual cells is also an essential enabling technique to move cellular biology, biomedicine, and biotechnology forward. Due to its great potential to solve bio-related problems, this cutting-edge technique has become a topic of concern for many studies on micro-manipulation methods. Hence, we present here a comprehensive review on recent developments of cancer cell separation utilizing various microfluidic-based methods, including dielectrophoresis-, magnetic-, acoustic-, and passive microfluidic-based ones. The review includes an overview of how these methods work along with a description of the experimental results using each method as well as their respective advantages and disadvantages. The paper is wrapped up with a discussion of the current challenges and future prospects in the cell separation technique. Our conclusion is that this technique will allow more opportunities for biomedical and bioengineering researchers to improve lab-on-a-chip technologies, and will have far-reaching implications for bio-related studies and applications.