Acanthamoeba keratitis is an aggressive and rapidly progressing ocular pathology whose main risk factor is the use of contact lenses. An early and differential diagnosis is considered the main factor to prevent the progression and improve the prognosis of the pathology. However, current diagnosis techniques require time, complex and costly materials making an early diagnosis challenging. Thus, there is a need for fast, accessible, and accurate methods for Acanthamoeba detection by practitioners for timely and suitable treatment and even for contact lens user as preventive diagnosis. Here, we developed a dual-mode colorimetric-based method for fast, visual, and accurate detection of Acanthamoeba using gold nanoparticles (AuNPs). For this strategy, AuNPs were functionalized with thiolated probes and the presence of target Acanthamoeba genomic sequences, produce a colorimetric change from red to purple. This approach allows the detection of 0.02 and 0.009 μM of the unamplified Acanthamoeba genome by the naked eye in less than 20 min and by color analysis using a smartphone. Additionally, real samples were successfully analyzed showing the potential of the technology considering the lack of point-of-care tools that are mostly needed.