Holographic optical tweezers (HOT) is a programmable technique used for manipulation of microsized samples. In combination with computer-generation holography (CGH), a spatial light modulator reshapes the light distribution within the focal area of the optical tweezers. HOT can be used to realize real-time multiple-point manipulation in fluid, and this is useful in biological research. In this article, we summarize the HOT technique, discuss its recent developments, and present an overview of its biological applications.