Abstract: In order to more reasonably analyze conservative sites and conservative regions in biological sequences, a new method of biological sequences comparison with constraints on element position is proposed. Based on theoretical analysis of the new method and the traditional method, it is shown that the complexity of the new method is the same as that of the traditional method, but the total amount of needed computations may decrease 1/3 to 2/3. Computing experiments show that the needed time for the new method may be 1/4 to 2/5 less than for the traditional method, and that the result computed by the new method is almost the same as that by the traditional method if the two sequences are comparable and the constraints are proper.