Pointing accuracy is a critical performance indicator of opto-mechanical systems, directly affecting the systems’ efficiency and application range. This study introduces what we believe to be a novel approach for predicting pointing accuracy and adjusting processes in opto-mechanical systems, considering multi-source uncertainty quantification. First, the relationship between error components and total error is quantified using homogeneous coordinate transformation theory. Second, by applying the Nataf transformation to uncertain variables, a hybrid interval-probabilistic uncertainty quantification model based on generalized polynomial chaos is constructed. Third, by selecting points from the probability distribution domain, a parameterized finite element simulation is conducted to create a pointing accuracy prediction model, obtaining the theoretical limit accuracy for the opto-mechanical system. Finally, considering multi-bolt elastic interactions, an assembly process adjustment model is developed to achieve performance-based assembly process adjustments, and tests are conducted to measure the pointing accuracy of the opto-mechanical system after calibration. Pointing accuracy measurements following calibration showed an improvement from 249” to 117”, an increase of 53.01%, approaching the theoretical limit of 108”. This approach requires only one adjustment to approach optimal accuracy compared to eight adjustments with traditional methods, greatly enhancing assembly efficiency. This study offers a theoretical foundation for predicting and adjusting pointing accuracy in opto-mechanical systems.