Laser speckle contrast imaging (LSCI) is a powerful tool for monitoring blood flow changes in tissue or vessels in vivo, but its applications are limited by shallow penetration depth under reflective imaging configuration. The traditional LSCI setup has been used in transmissive imaging for depth extension up to 2lt–3lt (lt is the transport mean free path), but the blood flow estimation is biased due to the depth uncertainty in large depth of field (DOF) images. In this study, we propose a transmissive multifocal LSCI for depth-resolved blood flow in thick tissue, further extending the transmissive LSCI for tissue thickness up to 12lt. The limited-DOF imaging system is applied to the multifocal acquisition, and the depth of the vessel is estimated using a robust visibility parameter Vr in the coherent domain. The accuracy and linearity of depth estimation are tested by Monte Carlo simulations. Based on the proposed method, the model of contrast analysis resolving the depth information is established and verified in a phantom experiment. We demonstrated its effectiveness in acquiring depth-resolved vessel structures and flow dynamics in in vivo imaging of chick embryos.