Fully lamellar (FL) Ti-46.5Al-2Cr-1.5Nb-1V (at%) alloy is used to study the relationship between microstructure and fracture toughness. A heat treatment process is adopted to control the microstructural parameters of the studied alloy. Fracture toughness experiments and scanning electron microscope (SEM) in-situ straining experiments are carried out to determine the influence of lamellar spacing and grain size on the fracture toughness of FL TiAl alloys. It is found that ligament length depends on the lamellar spacing, and fracture toughness varies non-monotonously with the increase of grain size. The results are ascribed to the competition between the microcrack nucleation and microcrack propagation. Finally a semi-empirical relationship between the fracture toughness and microstructure parameters was established.