Multiple-input multiple-output (MIMO) and orthogonal frequency division multiplexing (OFDM) are two key techniques for broad-band wireless mobile communications and channel state information (CSI) is critical for the realization and performance of MIMO-OFDM systems in doubly-selective fading channels. Channel estimation based on two-dimensional discrete-time Fourier transform interpolation (2D-DFTI) is a promising solution to obtain accurate CSI for MIMO-OFDM systems in theory because of both its robustness and high computational efficiency, however, its performance will degrade significantly in practical MIMO-OFDM systems due to the two-dimensional Gibbs phenomenon caused by virtual subcarriers and burst transmission. In this paper, we propose a novel channel estimation method based on the two-dimensional enhanced DFT interpolation (2D-EDFTI), i.e., the frequency-domain EDFTI (FD-EDFTI) concatenated with the time-domain EDFTI (TD-EDFTI), for practical burst-mode MIMO-OFDM systems with virtual subcarriers, which can increase the channel estimation accuracy effectively by mitigating the Gibbs phenomenon in frequency-domain and time-domain, respectively, while keeping good robustness and high computational efficiency. In addition to computer simulations, we further implement the 2D-EDFTI channel estimator into our real-time FPGA testbed of 4 x 4 MIMO-OFDM transmission via spatial multiplexing, together with different MIMO detectors. Both computer simulations and RF experiments demonstrate the superior performance of 2D-EDFTI channel estimation in doubly-selective fading channels, therefore, high-throughput MIMO-OFDM transmission based on different MIMO detection algorithms can always be well supported. Also, it can be applied to other MIMO-OFDM transmission schemes straightforwardly.