Background Establishing the underlying cause of anaemia in general practice is a diagnostic challenge. Currently, general practitioners individually determine which laboratory tests to request (routine work-up) in order to diagnose the underlying cause. However, an extensive work-up (consisting of 14 tests) increases the proportion of patients correctly diagnosed. This study investigates the cost-effectiveness of this extensive work-up. Methods A decision-analytic model was developed, incorporating all societal costs from the moment a patient presents to a general practitioner with symptoms suggestive of anaemia (aged ≥ 50 years), until the patient was (correctly) diagnosed and treated in primary care, or referred to (and diagnosed in) secondary care. Model inputs were derived from an online survey among general practitioners, expert estimates and published data. The primary outcome measure was expressed as incremental cost per additional patient diagnosed with the correct underlying cause of anaemia in either work-up. Results The probability of general practitioners diagnosing the correct underlying cause increased from 49.6% (95% CI: 44.8% to 54.5%) in the routine work-up to 56.0% (95% CI: 51.2% to 60.8%) in the extensive work-up (i.e. +6.4% [95% CI: −0.6% to 13.1%]). Costs are expected to increase slightly from €842/patient (95% CI: €704 to €994) to €845/patient (95% CI: €711 to €994), i.e. +€3/patient (95% CI: €−35 to €40) in the extensive work-up, indicating incremental costs of €43 per additional patient correctly diagnosed. Conclusions The extensive laboratory work-up is more effective for diagnosing the underlying cause of anaemia by general practitioners, at a minimal increase in costs. As accompanying benefits in terms of quality of life and reduced productivity losses could not be captured in this analysis, the extensive work-up is likely cost-effective.