As tuberculosis (TB) still claims millions of lives in the world, more research should be directed toward understanding this disease and finding a more effective solution. In vivo human alveolar macrophages are an important focus in TB research since the causative agent, Mycobacterium tuberculosis, can be harbored within these cells in an inactive form for a long time before reactivation. To date, TB researchers collect alveolar macrophage samples using a special clinical procedure known as bronchoalveolar lavage (BAL) in which a bronchoscope is inserted into the lung and the cell samples are collected after being washed out with fluid. BAL is an invasive procedure and its use for a clinical study would often meet with practical constraints and the patient’s psychological resistance. These concerns led us to seek a non-invasive approach. In the present study, we collected alveolar macrophage samples from patients with active tuberculosis using a simple sputum expectoration procedure and used the samples to assess the gene expression activities of these immune cells. In this work, we made contributions in two aspects. This study is the first to demonstrate that sputum expectoration is a useful non-invasive alternative for collecting alveolar macrophages whose gene activities would serve to monitor the disease activity. In addition, we analyzed the gene expression of alveolar macrophages in the in vivo samples obtained from TB patients, in contrast to related work where gene expression was profiled on alveolar macrophages collected from non-TB patients and then infected with Mycobacterium tuberculosis in vitro.