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Modeling the relationship between vehicle speed and density on the road is a fundamental problem in traffic flow theory. Recent research found that using the least-squares (LS) method to calibrate single-regime speed-density models is biased because of the uneven distribution of samples. This paper explains the issue of the LS method from a statistical perspective: the biased calibration is caused by the correlations/dependencies in regression residuals. Based on this explanation, we propose a new calibration method for single-regime speed-density models by modeling the covariance of residuals via a zero-mean Gaussian Process (GP). Our approach can be viewed as a generalized least-squares (GLS) method with a specific covariance structure (i.e., kernel function) and is a generalization of the existing LS and the weighted least-squares (WLS) methods. Next, we use a sparse approximation to address the scalability issue of GPs and apply a Markov chain Monte Carlo (MCMC) sampling scheme to obtain the posterior distributions of the parameters for speed-density models and the hyperparameters (i.e., length scale and variance) of the GP kernel. Finally, we calibrate six well-known single-regime speed-density models with the proposed method. Results show that the proposed GP-based methods (1) significantly reduce the biases in the LS calibration, (2) achieve a similar effect as the WLS method, (3) can be used as a non-parametric speed-density model, and (4) provide a Bayesian solution to estimate posterior distributions of parameters and speed-density functions.