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<i>Euglena gracilis</i> (<i>E. gracilis</i>) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of <i>E. gracilis</i> mediated by <i>Agrobacterium</i> (<i>A. tumefaciens</i>). <i>E. gracilis</i> was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. <i>A. tumefaciens</i>-mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the <i>gus</i> gene (encoding β-glucuronidase (GUS)) were found to be GUS-negative, indicating that the <i>gus</i> gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene (<i>hptII</i>) (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of <i>Euglena</i> with the <i>hptII-2A–gus</i> gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when <i>A. tumefaciens</i> LBA4404 (A₆₀₀ = 1.0) and <i>E. gracilis</i> (A₇₅₀ = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer <i>E. gracilis</i> for producing high-value products and fundamental studies.