A method and apparatus for transmitting a preamble for frame synchronization and channel estimation in a MIMO-OFDM communication system are provided. An OFDM communication system using Q transmit antennas generates a base preamble sequence including a CP and an orthogonal sequence. If Q≦a predetermined number M, a preamble sequence for a kth antenna is S(t−(k−1)T/M). If Q>M and k≦M, the preamble sequence transmitted for the kth antenna is S(t−(k−1)T/M). If Q>M and k>M, the preamble sequence for the kth antenna is (−1)(PS-1)S(t−(k−M−1)T/M). Here, S(t) is the orthogonal sequence, T is the period of the orthogonal sequence, and PS is an index indicating a transmission period of the preamble sequence. The preamble sequences are at least twice transmitted from the Q transmit antennas.