Miniaturization of biosensors is envisaged by the development of biochips consisting of parallel microarray patterns of binding sites on rigid substrates, such as glass or silicon. Thin plastic substrates are promising flexible alternatives because of the possibility for large-area roll-to-roll manufacturing of disposable chips at lower costs. Mature optical lithography technology faces many challenges when used to pattern flexible foils as a result of the substrate instabilities, especially at higher temperatures. In this work, flexible biochips with gold electrode patterns were fabricated on thin polyethylene naphthalate (PEN) foils using photolithography. The gold electrode structures of the chips were manufactured by direct metal patterning and by lift-off processing. Both methodologies resulted in well-defined electrode patterns as concluded from optical microscopy and scanning electron microscopy (SEM) characterization and resistance measurements. The biochips were successfully employed for the electrical and optical detection of DNA molecules. The DNA detection was based on the immobilization of capture DNA between electrode gaps, hybridization with biotin-labeled target DNA, and enzymatic silver enhancement.