Optothermal tweezers (OTT) have emerged as a transformative technology in nanotechnology and life sciences, addressing the limitations of traditional optical tweezers, such as the diffraction barrier and thermal damage to samples. By coupling light and heat to create precise temperature gradients, OTT generate thermophoretic forces to manipulate particles with spatial resolution beyond the diffraction limit and at power densities significantly lower than conventional optical tweezers. This capability enables the trapping of delicate biological samples and nanoscale particles, including biomolecules, metallic nanoparticles, and polymeric materials, with minimal optical heating damage. OTT’s versatility extends to large-scale particle assembly, facilitating the creation of complex nanostructures and biomimetic systems, and their applications span diverse fields, including targeted drug delivery, biosensing, synthetic biology, and single-cell manipulation. Recent advancements, such as the integration of low-temperature environments and opto-refrigerative designs, further enhance their precision and adaptability. This chapter systematically explores the principles of OTT, their biomedical and nanotechnological applications, and their transformative potential across disciplines. Additionally, we address the challenges in temperature gradient control, thermal effects on sensitive systems, and scalability for industrial applications. By overcoming these barriers through interdisciplinary efforts and innovative engineering, OTT are poised to revolutionize micro- and nanoscale manipulation, paving the way for unprecedented scientific and technological advancements.