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The MIMO-OFDM (multiple input multiple output-orthogonal frequency division multiplexing) system recently received considerable attention because of its potential ability to enable the higher transmission data rate while keeping the high signal quality. In practical MIMO-OFDM systems, the efficient and highly accurate channel estimation method is the mandatory requirement to achieve the potential system performance over MIMO channels. This paper proposes the discrete cosine transform (DCT) interpolation-based channel estimation (DCTI-CE) method, which could meet the above requirements. The salient feature of the proposed DCTI-CE method is to enable the estimation of all link channel responses with high accuracy by using only one preamble symbol even under the severe multi-path fading channel for wireless LAN systems.