I would suggest to play with this example. http://nilearn.github.io/auto_examples/04_manipulating_images/plot_nifti_simple.html#sphx-glr-auto-examples-04-manipulating-images-plot-nifti-simple-py
NiftiMasker has an argument called mask_img.
Initialize the object
masker = NiftiMasker(mask_img=nifti_mask)
Use for loop to extract values from ROI on each subject.
signals = masker.fit_transform(img)
HTH