Affective state can influence cognition leading to biased information processing, interpretation, attention, and memory. Such bias has been reported to be essential for the onset and maintenance of different psychopathologies, particularly affective disorders. However, empirical evidence has been very heterogeneous and little is known about the neurophysiological mechanisms underlying cognitive bias and its time-course. We therefore investigated the interpretation of ambiguous stimuli as indicators of biased information processing with an ambiguous cue-conditioning paradigm. In an acquisition phase, participants learned to discriminate two tones of different frequency, which acquired emotional and motivational value due to subsequent feedback (monetary gain or avoidance of monetary loss). In the test phase, three additional tones of intermediate frequencies were presented, whose interpretation as positive (approach of reward) or negative (avoidance of punishment), indicated by a button press, was used as an indicator of the bias. Twenty healthy volunteers participated in this paradigm while a 64-channel electroencephalogram was recorded. Participants also completed questionnaires assessing individual differences in depression and rumination. Overall, we found a small positive bias, which correlated negatively with reflective pondering, a type of rumination. As expected, reaction times were increased for intermediate tones. ERP amplitudes between 300 - 700 ms post-stimulus differed depending on the interpretation of the intermediate tones. A negative compared to a positive interpretation led to an amplitude increase over frontal electrodes. Our study provides evidence that in humans, as in animal research, the ambiguous cue-conditioning paradigm is a valid procedure for indirectly assessing ambiguous cue interpretation and a potential interpretation bias, which is sensitive to individual differences in affect-related traits.