We define the counting of holomorphic cylinders in log Calabi-Yau surfaces. Although we start with a complex log Calabi-Yau surface, the counting is achieved by applying methods from non-archimedean geometry. This gives rise to new geometric invariants. We prove that the counting satisfies a property of symmetry. Explicit calculations are given for a del Pezzo surface in detail, which verify the conjectured wall-crossing formula for the focus-focus singularity. Our holomorphic cylinders are expected to give a geometric understanding of the combinatorial notion of broken line by Gross, Hacking, Keel and Siebert. Our tools include Berkovich spaces, tropical geometry, Gromov-Witten theory and the GAGA theorem for non-archimedean analytic stacks.