The nondestructive reconstruction of three-dimensional (3D) temperature field in biological tissue is always an important problem to be resolved in biomedical engineering field. This paper presents a novel method of nondestructive reconstruction of 3D temperature field in biological tissue based on multi-island genetic algorithm (MIGA). By this method, the resolving of inverse problem of bio-heat transfer is transformed to be a solving process of direct problem. An experiment and its corresponding simulation were carried out to verify the feasibility and reliability. In the experiment a high purity polypropylene material, whose thermophysical parameters were similar to the fat tissue being tested, were adopted so that it could avoid the negative results created by the other factors. We set the position P(x, y, z) as the point heat source in the biological tissue and its temperature t as optimization variable, got the experimental temperature values of the points in a module surface, subtracted them from the corresponding simulating temperature values in the same module surface, and then took the sum of absolute value. We took it as the objective function of successive iteration. It was found that the less the target value was, the more optimal the current variables, i.e. the heat source position and the temperature values, were. To improve the optimization efficiency, a novel establishment method of objective function was also provided. The simulating position and experimental position of heat source were very approximate to each other. When the optimum values are determined, the corresponding 3D temperature field is also confirmed, and the temperature distribution of arbitrary section can be acquired. The MIGA can be well applied in the reconstruction of 3D temperature field in biological tissue. Because of the differences between the MIGA and the traditional numerical methods, we do not have to acquire all the data of surface. It is convenient and fast, and shows a prosperous application future.