The operating comfort of biological microscopes is directly related to the operators’ health and the accuracy of observation results. In this paper, the neck and arm comfort of biological microscope operators was researched. Firstly, the evaluation experiments included two parts: one was the evaluation of operating comfort for the neck and shoulder, the other was for the arm. The surface electromyography (sEMG) data of the measured muscles such as the splenius capitis, sternocleidomastoid, trapezius, flexor carpi and brachioradialis were acquired within the range of 0–40° neck angle and 90–150° elbow angle. In addition, the rating of perceived exertion (RPE) of operators was taken as the subjective evaluation results for different operating postures. Secondly, the root mean square (RMS) data of different subjects’ sEMG were normalized to the corresponding RMS of 20%MVC. It can be seen that the load of the splenius capitis, sternocleidomastoid and trapezius all increased with the increase of the head bow angle, and the load of measured muscles reached the maximum in 35–45°. And when the angle between the forearm and the upper arm was 115–125°, the load on the flexor carpi radialis, brachioradialis and trapezius was minimal. Finally, the ANOVA statistical analysis was proceeded to the RMS data of the measured muscles. It was found that the load of the splenius capitis and sternocleidomastoid was positively correlated with the bow angle, and the load range was 4%–30%MVC.