We present a novel bifocal imaging method enabling three-dimensional particle tracking and size determination employing a single camera only. The method is based on double refraction causing a particle to be imaged twice, each particle image of different blur. From these double images, a linear calibration function can be derived allowing to determine the three-dimensional particle position unambiguously over the entire depth of measurement volume. As this calibration function is independent of the particle size used, the particle size can be determined simultaneously by relating size of the double images and depth position of the particle. To prove the applicability, a co-laminar flow of two particle suspensions with particles of 1.14 μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}m and 2.47 μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}m in diameter was measured in a Y-shaped microchannel. While the laminar flow field was measured with very low uncertainty and independent of the particle size, the particle size distributions determined reproduced reliably the size distributions expected for the co-laminar flow applied, with a precision of about 98.6 %\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\%$$\\end{document} regarding the particle size discrimination. The progress for research is a new method readily to implement in common optical setups, promising, for example, valuable insights in polydisperse suspension flows—the vast majority of flows in fundamental research and applications.Graphical abstract