Scanning electrochemical Microscopy (SECM) has been used to noninvasively characterize oxygen consumption rate of single mammalian embryos and oocytes under physiological condition in culure medium at 37 °C. Local oxygen concentration profile near the embryo sample was monitored by scanning with a Pt microelectrode probe, and then mass transfer rate for oxygen has been estimated based on spherical diffusion theory. A bovine embryo at two-cell stage was located in either a conventional culture dish or a cone-shaped microwell and compared the differences in concentration profile and diffusion behavior. We found that the cone-shaped microwell functions to amplify the oxygen concentration difference between the sample surface and the bulk. Further more, a measuring plate equipped with the cone-shaped six-microwells was developed to easily handle many embryos in a short time. The respiration activities significantly increased with the embryo development for both bovine and mouse.