We developed optically driven microtools for processing single biomolecules using a microfluidic workbench composed of a microfluidic platform that functions under an optical microscope. The optically driven microtools have enzymes immobilized on their surfaces, which catalyze chemical reactions for molecular processing in a confined space. Optical manipulation of the microtools enables them to be integrated with a microfluidic device for controlling the position, orientation, shape of the target sample. Here, we describe the immobilization of enzymes on the surface of microtools, the microfluidics workbench, including its microtool storage and sample positioning functions, and the use of this system for on-site cutting of single chromosomal DNA molecules. We fabricated microtools by UV lithography with SU-8 and selected ozone treatments for immobilizing enzymes. The microfluidic workbench has tool-stock chambers for tool storage and micropillars to trap and extend single chromosomal DNA molecules. The DNA cutting enzymes DNaseI and DNaseII were immobilized on microtools that were manipulated using optical tweezers. The DNaseI tool shows reliable cutting for on-site processing. This pinpoint processing provides an approach for analyzing chromosomal DNA at the single-molecule level. The flexibility of the microtool design allows for processing of various samples, including biomolecules and single cells.