A method for three-dimensional microdissection for separating defined structures in the sub-millimeter range by cold laser ablation or multi-photon absorption, whereby exposure of the structures to be separated is performed using directional information in all spatial directions. Also, a 3D microdissection system for separating defined, three-dimensional structures from a sample, having: a control unit (5); an ablation chamber (1) with a sample holder, on which the sample to be processed is mounted and which is movable along a linear axis V and rotatable about a rotary axis R. The sample holder has positioning devices connected to the control unit. The positioning devices move the sample holder along another linear axis H and rotate it about rotary axis R, and a laser device (6) is introduced into the ablation chamber at least partially through a laser window (3) in the ablation chamber. The ablation chamber is connected to the control unit and has an adjustable optic, such that the laser beam is focused near the sample.