The retinal nerve fiber layer (RNFL) evaluation is becoming a very effective method for the clinical diagnosis of early glaucoma. The purpose of this paper is to extract the pulsations of the RNFL, which might be used as a novel biomarker for glaucoma diagnosis. To demonstrate that the optical coherence tomography (OCT) could extract the subtle RNFL dynamic pulsatile motion in normal eyes in vivo, the subjects’ retina was imaged by spectral domain optical coherence tomography (SD-OCT) based on histogram RNFL pulse extraction algorithm. Firstly, B-scan images of multiple retinal layers in normal subjects were acquired. The RNFL was identified from each B-scan with a segmentation algorithm based on shortest path and convolutional neural network. Secondly, a histogram-based RNFL pulsation extraction algorithm was proposed to track the displacement of the RNFL which is based on the acquired RNFL B-scan images. Finally, in evaluating the dynamic pulse signal extracted from the pulsating motion of RNFL, an experiment was designed to collect heart rate using an infrared pulse sensor device. The cardiac pulse waveform and the RNFL pulse waveform were compared and analyzed in time and frequency domain. The results show that the extracted RNFL pulse has the same frequency as the cardiac pulse, which validate the feasibility and accuracy of the in vivo extraction scheme used in this paper.