Three-dimensionally ordered macroporous (3DOM) polytetrafluoroethylene (PTFE) has hardly been prepared due to the poor solubility in most of solvents and high melting temperature of PTFE raw material. In this work, monodispersed polystyrene (PS) microspheres and PTFE latex particles were controlled to simultaneously deposit from water. After the deposits were calcinated at 400 °C, a self-supported superhydrophobic 3DOM PTFE film with a static water contact angle of 154° was successfully fabricated. The pore size can be tunable from 1.5 to 3 μm, depending on the size of PS microspheres. The prepared 3DOM PTFE film were used as micromolds and microreactors to prepare poly(methyl methacrylate) (PMMA) particles and TiO2 macroporous material, respectively. This work provides a facile method to fabricate 3DOM PTFE materials.