The single beam optical trap (optical tweezers), a highly focused beam, is on its way to revolutionizing not only the fields of colloidal physics and biology, but also materials science and engineering. Recently, spatially-extended three-dimensional light patterns have gained considerable usage for exerting force to alter, manipulate, organize and characterize materials. To advance the degree of manipulation, such as rotation of materials in microfluidic environments along with spatial structuring, other beam parameters such as phase and polarization have to be configured. These advances in optical tweezers’ technology have enabled complex microfluidic actuation and sorting. In addition to remotely (in a non-contact way) applying force and torques in three-dimensions, which can be continuously varied unlike mechanical manipulators, optical tweezers-based methods can be used for sensing the force of interaction between microscopic objects in a microfluidic environment and for the characterization of micro-rheological properties. In this review, we place emphasis on applications of optical actuation based on novel beams in performing special functions such as rotation, transportation, sorting and characterization of the microscopic objects. Further, we have an extended discussion on optical actuation (transport and rotation) with fiber optic microbeams and spectroscopic characterization in the microfluidic environment. All these advancements in optical manipulation would further facilitate the growing use of optical tools for complex microfluidic manipulations.