A couple functions for generating wav files with a 3 byte sample width.

create_sample24.py

# create_sample24.py
# Author: Warren Weckesser
# License: BSD 3-Clause (http://opensource.org/licenses/BSD-3-Clause)

import numpy as np
import wave


def create_sample():
    # 27 bytes (1 channel, 9 frames, 3 bytes per frame)
    # The bytes are simply 1, 2, 3, ..., 27.
    data = ''.join(chr(k) for k in range(1, 28))

    w = wave.open('sample24.wav', 'wb')
    w.setnchannels(1)
    w.setsampwidth(3)
    w.setframerate(22050)
    w.writeframes(data)
    w.close()


def create_sine():
    # Create a 24 bit wav file containing a pure sine wave.

    rate = 22050  # samples per second
    T = 3         # sample duration (seconds)
    f = 440.0     # sound frequency (Hz)

    t = np.linspace(0, T, T*rate, endpoint=False)
    x = (2**23 - 1) * np.sin(2 * np.pi * f * t)
    a = x.astype(np.int32).view(np.uint32)
    # By shifting first 0 bits, then 8, then 16, the resulting output
    # is 24 bit little-endian.
    b = (a.reshape(-1, 1) >> np.array([0, 8, 16])) & 255
    data = b.astype(np.uint8).tostring()

    w = wave.open('sine24.wav', 'wb')
    w.setnchannels(1)
    w.setsampwidth(3)
    w.setframerate(rate)
    w.writeframes(data)
    w.close()


if __name__ == "__main__":
    create_sample()
    create_sine()