An optical scanning observation apparatus having an internal focusing mechanism. suitable for in-vivo observation of animals, is provided. The optical scanning observation apparatus includes a light source, an objective optical system for focusing light onto the sample, a detection-light splitting device for splitting off collected detection light, a detector for detecting the focused detection light, a focus scanning device disposed between the detection-light splitting device and the objective optical system, and a lateral-direction scanning device, disposed between the focus scanning device and the objective optical system, for scanning the light in orthogonal directions relative to the optical axis. The focus scanning device further includes a focusing optical system having a positive and negative lens group and a lens driving device for moving one lens group to adjust the working distance of the objective optical system.