A novel uncertainty-based pressure reconstruction method is proposed to evaluate the instantaneous pressure fields from velocity fields measured using particle image velocimetry (PIV) or particle tracking velocimetry (PTV). First, the pressure gradient fields are calculated from velocity fields, while the local and instantaneous pressure gradient uncertainty is estimated from the velocity uncertainty using a linear-transformation based algorithm. The pressure field is then reconstructed by solving an overdetermined linear system which involves the pressure gradients and boundary conditions. This linear system is solved with generalized least-squares (GLS) which incorporates the previously estimated variances and covariances of the pressure gradient errors as inverse weights to optimize the reconstructed pressure field. The method was validated with synthetic velocity fields of a 2D pulsatile flow and the results show significantly improved pressure accuracy with an error reduction of as much as 250% compared to the existing baseline method of solving the pressure Poisson equation (PPE). The GLS was more robust to the velocity errors and provides greater improvement with spatially correlated velocity errors. For experimental validation, the volumetric pressure fields were evaluated from a laminar pipe flow velocity field measured using 3D PTV. The GLS reduced the median absolute pressure errors by as much as 96%.