Differential item functioning (DIF), referring to between-group variation in item characteristics above and beyond the group-level disparity in the latent variable of interest, has long been regarded as an important item-level diagnostic. The presence of DIF impairs the fit of the single-group item response model being used, and calls for either model modification or item deletion in practice, depending on the mode of analysis. Methods for testing DIF with continuous covariates, rather than categorical grouping variables, have been developed; however, they are restrictive in parametric forms, and thus are not sufficiently flexible to describe complex interaction among latent variables and covariates. In the current study, we formulate the probability of endorsing each test item as a general bivariate function of a unidimensional latent trait and a single covariate, which is then approximated by a two-dimensional smoothing spline. The accuracy and precision of the proposed procedure is evaluated via Monte Carlo simulations. If anchor items are available, we proposed an extended model that simultaneously estimates item characteristic functions (ICFs) for anchor items, ICFs conditional on the covariate for non-anchor items, and the latent variable density conditional on the covariate—all using regression splines. A permutation DIF test is developed, and its performance is compared to the conventional parametric approach in a simulation study. We also illustrate the proposed semiparametric DIF testing procedure with an empirical example.