Anti-synthetase syndrome (ASS) is a rare autoimmune disorder characterized by interstitial lung disease (ILD), myositis, and arthritis, primarily associated with antibodies, such as anti-Jo-1, that target the t-ribonucleic acid (tRNA) synthetase enzymes. This case report describes a 45-year-old man who presented with isolated flexor tenosynovitis and bilateral hand pain, later diagnosed with anti-Jo-1 positive ASS. Initially, the patient's symptoms were attributed to a trivial injury, but subsequent imaging and laboratory evaluations revealed tenosynovitis without signs of inflammatory arthritis. Positive anti-Jo-1 antibodies and later elevated creatine kinase (CK) levels indicated the development of myositis, supported by magnetic resonance imaging (MRI) findings and muscle biopsies showing mild inflammatory features. The patient was treated with prednisone and mycophenolate mofetil, resulting in significant clinical improvement and resolution of symptoms. This case highlights an atypical presentation of ASS, with isolated tenosynovitis as an initial symptom, which is not commonly documented in the literature. The findings underscore the importance of considering ASS in patients with unexplained inflammatory symptoms and highlight the role of comprehensive clinical evaluation and antibody testing in guiding diagnosis. By recognizing the variable clinical manifestations of ASS, clinicians can facilitate earlier diagnosis and prompt treatment, improving patient outcomes.