DTW measures is an indication of dissimilarity, which has a wide range of applications in pattern recognition and machine learning. Nevertheless, it is difficult to directly calculate the distance between unequal-sized matrices, which can potentially be solved by using the proposed Non-Interlaced Dynamic Time Warping (Non-Interlaced DTW) algorithm in this paper. In the new algorithm, matrices are separated into row vectors, and DTW distance matrix is set up based on the distances between row vectors. By introducing dynamic programming, the minimum cumulative distance and shortest path of the DTW distance matrix can be achieved, as well as the optimal match and the rows-alignment between row vectors. Non-Interlaced DTW algorithm defines the normalized cumulative distance of DTW distance matrix as the final distance measure. The performance of the algorithm is tested and verified by experiments on MNIST database, FVC2004 dataset and Caltech database. The results illustrate that the Non-Interlaced DTW can get accurate similarity between unequal-sized matrixes. By directly obtaining the distance between matrices, Non-Interlaced DTW provides a new idea of similarity measure for unequal-sized matrices and has the promising application when dealing with high-dimensional data.