An unbiased algorithm of generalized linear least squares (GLLS) for parameter estimation of nonuniformly sampled biomedical systems is proposed. The basic theory and detailed derivation of the algorithm are given. This algorithm removes the initial values required and computational burden of nonlinear least regression and achieves a comparable estimation quality in terms of the estimates' bias and standard deviation. Therefore, this algorithm is particular useful in image-wide (pixel-by-pixel based) parameter estimation, e.g., to generate parametric images from tracer dynamic studies with positron emission tomography. An example is presented to demonstrate the performance of this new technique. This algorithm is also generally applicable to other continuous system parameter estimation.