When confocal microscopy is combined with resonance Raman spectroscopy, the focusing accuracy of the system decreases due to the movement of the convergence plane of the beam caused by the change of the working wavelength. In this paper, a differential confocal microscopy focusing technique with variable working wavelength is proposed, and the basic theory of confocal technique based on the wavelength-convergence plane position relationship is established, which can provide a precise theoretical basis for the determination of the focusing accuracy of the confocal resonance Raman technique, and the axial resolving power is 6 nm, which greatly improves the system accuracy. It is experimentally verified that this technique can eliminate the influence of the change of working wavelength on the focusing accuracy of the system, which lays the foundation for detecting the morphology imaging of micro-regions, and provides theoretical support and help for the development of the confocal resonance Raman technique.