Cognitive affective biases describe the tendency to process negative information or positive information over the other. These biases can be modulated by changing extracellular serotonin (5-HT) levels in the brain, for example, by pharmacologically blocking and downregulating the 5-HT transporter (5-HTT), which remediates negative affective bias. This suggests that higher levels of 5-HTT are linked to a priority of negative information over positive, but this link remains to be tested in vivo in healthy individuals. We, therefore, evaluated the association between 5-HTT levels, as measured with [¹¹C]DASB positron emission tomography (PET), and affective biases, hypothesising that higher 5-HTT levels are associated with a more negative bias. We included 98 healthy individuals with measures of [¹¹C]DASB binding potential (BP_ND) and affective biases using The Emotional Faces Identification Task by subtracting the per cent hit rate for happy from that of sad faces (EFIT_AB). We evaluated the association between [¹¹C]DASB BP_ND and EFIT_AB in a linear latent variable model, with the latent variable (5-HTT_LV) modelled from [¹¹C]DASB BP_ND in the fronto-striatal and fronto-limbic networks implicated in affective cognition. We observed an inverse association between 5-HTT_LV and EFIT_AB (β = −8% EFIT_AB per unit 5-HTT_LV, CI = −14% to −3%, p =.002). These findings show that higher 5-HTT levels are linked to a more negative bias in healthy individuals. High 5-HTT supposedly leads to high clearance of 5-HT, and thus, a negative bias could result from low extracellular 5-HT. Future studies must reveal if a similar inverse association exists in individuals with affective disorders.