When a muscle fiber direction and an electrode direction are different, some problems will occur in the surface electromyogram measurement. Therefore, by this study, we propose the surface electromyogram measurement procedure that a muscle fiber direction can estimate from data after measurement. This measurement procedure records differential signals between a reference electrode signal and a signal from all electrodes of the matrix electrode. We can obtain the differential signal between any electrodes by the calculation. We placed a matrix electrode and reference electrode on the biceps brachii muscle and recorded surface electromyogram. By this measurement procedure, we were able to estimate the muscle fiber direction from the data after the measurement. We can also choose two electrodes which do not sandwich the neuromuscular junction and along a muscle fiber direction from a matrix electrode.