Abstract: A three-dimensional detection method for red tide algae based on holographic imaging is provided, including: constructing a coaxial optical path based on a structure of a Mach-Zehnder interferometer; determining reconstruction distances of the system under different object distances; constructing an original hologram database of the algae; selecting an original hologram, and two reconstructed holograms with different reconstruction distances as a three-dimensional spatial sample of the original hologram; labeling algae in sample, and recording species and bounding boxes of the algae; training a target detection network with the sample of the holograms and a label file; detecting, with the trained target detection neural network, the sample of the algae to obtain species and a quantity of plankton in imaged water. The disclosure further provides a three-dimensional detection device for red tide algae. The method can improve a detection speed and detection accuracy of the red tide algae.