3D printing, an additive manufacturing process, draws particular attention due to its ability to produce components directly from a 3D model; however, the mechanical properties of the produced pieces are limited. In this paper, we present, from the experimental aspect, the fatigue behavior and damage analysis of polylactic acid (PLA)-Graphene manufactured using 3D printing. The main purpose of this paper is to analyze the combined effect of process parameters, loading amplitude, and frequency on fatigue behavior of the 3D-printed PLA-Graphene specimens. Firstly, a specific case study (single printed filament) was analyzed and compared with spool material for understanding the nature of 3D printing of the material. Specific experiments of quasi-static tensile tests are performed. A strong variation of fatigue strength as a function of the loading amplitude, frequency, and process parameters is also presented. The obtained experimental results highlight that fatigue lifetime clearly depends on the process parameters as well as the loading amplitude and frequency. Moreover, when the frequency is 80 Hz, the coupling effect of thermal and mechanical fatigue causes self-heating, which decreases the fatigue lifetime. This paper comprises useful data regarding the mechanical behavior and fatigue lifetime of 3D-printed PLA-Graphene specimens. In fact, it evaluates the effect of process parameters based on the nature of this process, which is classified as a thermally-driven process.