In　robotic　micromanipulation　systems,　the　orthogonality　of　the　three-axis　motion　trajectories　of　the　motion　control　systems　influences　the　accuracy　of　micromanipulation.　A　method　of　measuring　and　evaluating　the　orthogonality　of　three-axis　motion　trajectories　is　proposed　in　this　paper.　Firstly,　a　system　for　three-axis　motion　trajectory　measurement　is　developed　and　an　orthogonal　reference　coordinate　system　is　designed.　The　influence　of　the　assembly　error　of　laser　displacement　sensors　on　the　reference　coordinate　system　is　analyzed　using　simulation.　An　approach　to　estimating　the　orthogonality　of　three-axis　motion　trajectories　and　to　compensating　for　its　error　is　presented　using　spatial　line　fitting　and　vector　operation.　The　simulation　results　show　that　when　the　assembly　angle　of　the　laser　displacement　sensors　is　limited　within　a　range　of　10°,　the　relative　angle　deviation　of　the　coordinate　axes　of　the　reference　coordinate　frame　is　approximately　0.09%.　The　experiment　results　show　that　precision　of　spatial　line　fitting　is　approximately　0.02　mm　and　relative　error　of　the　orthogonality　measurement　is　approximately　0.3%.